Nsp molecules

ABSTRACT

The present invention relates to nucleotide sequences, including expressed sequence tags (ESTs), oligonucleotide probes, polypeptides, antagonists and agonists vectors and host cells expressing, and immunoadhesions and antibodies to Nsp1, Nsp2 and Nsp3.

This is a divisional of application Ser. No. 09/065,275 filed on Apr.23, 1998, which application is incorporated herein by reference and towhich application priority is claimed under 35 USC §120.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides which are characterized by the presence of novelSH2-containing proteins (Nsp's).

BACKGROUND OF THE INVENTION

Interactions between ligands and the cognate cell surface receptors arecritical for a variety of biological processes including maintenance ofcellular and organism homeostasis, development, and tumorigenesis. Manyof these ligands can activate multiple independent pathways and thestrength of the activation of different pathways can be modulated by thepresence or absence of signals generated by other receptors,Hotamisligil, et al., Proc. Nat. Acad. Sci. USA 91: 4854-58 (1994);Kanety et al., J. Biol. Chem. 270: 23780-84 (1995); Luttrell et al, J.Biol. Chem. 272: 4637-44 (1997). Adaptor molecules may be critical inintegrating multiple signaling cascades and in determining the cell typespecific response to extracellular stimuli. These adaptor proteins haveno apparent catalytic activity. Rather, they contain one or more domainsthat mediate protein-protein or protein-lipid interactions. The mostcommon conserved interaction domains in these adaptor molecules are Srchomology (SH2), SH3, phosphotyrosine binding (PTB) and pleckstrinhomology domains. [Reviewed in Pawson and Scott, Science 278: 2075-80(1997)].

Signals generated by growth factors such as epidermal growth factor(EGF) or insulin growth factor-1 (IGF-1) through receptor tyrosinekinases (RTK) or by extracellular matrix components acting through theintegrin receptors can induce cytoskeletal changes, Leventhal, el al.,J. Biol. Chem. 272: 5214-18 (1997); Ojaniemi & Vuori, J Biol. Chem. 272:25993-25998 (1996). There are also indications that RTKs can modulateintegrin signals and vice versa, Doerr & Jones, J. Biol. Chem. 271:2443-47 (1996); Jones et al., Proc. Natl. Acad Sci. USA 93: 2482-87(1996); Knight et al., J. Biol. Chem. 270: 10199-203 (1995); Matsumotoet al., Cancer Metas. Rev. 14: 205-17 (1995). However the details of howRTKs signal to the cytoskeletal components have not been fully resolved.Further, while some adaptor proteins have a limited pattern ofexpression [Liu & Roth, Proc. Natl. Acad. Sci. USA 92: 10287-91 (1995);Nakamura et al., Oncogene 13: 1111-21 (1996)], many are ubiquitouslyexpressed [Araki et al., Diabetes 42: 1041-54 (1993); Frantz et al., JBio. Chem. 272: 2659-67 (1997)]. Thus, it is not clear how biologicallyrelevant outputs are modulated as cells differentiate.

SUMMARY OF THE INVENTION

Applicants have identified cDNA clone (DNA30676 (SEQ ID NO:2), DNA40575(SEQ ID NO:4), DNA61601(SEQ ID NO:6)) that encodes a novel polypeptide,designated in the present application as "PRO201, PRO 308, PRO309,"respectively.

In one embodiment, the invention provides an isolated nucleic acidmolecule having at least about 80% sequence identity to (a) a DNAmolecule encoding a polypeptide comprising the sequence of amino acids 1to 576 of FIG. 1 (SEQ ID NO: 1), amino acids 1 to 501 of FIG. 2 (SEQ IDNO: 3) or amino acids 1 to 703 of FIG. 3 (SEQ ID NO: 5); or (b) thecomplement of the DNA molecule of (a). The sequence identity preferablyis about 85%, more preferably about 90%, most preferably about 95%. Inone aspect, the isolated nucleic acid has at least about 80%, preferablyat least about 85%, more preferably at least about 90%, and mostpreferably at least about 95% sequence identity with a polypeptidehaving amino acid residues 1 to 576 of FIG. 1 (SEQ ID NO: 1), 1 to 501of FIG. 2, (SEQ ID NO: 3), and 1 to 703 of FIG. 3 (SEQ ID NO: 5).Preferably, the greatest degree of identity occurs in the serine/prolinerich domain (i.e., amino acid residues 145-299 of SEQ ID NO: 1, aminoacid residues 28-210 of SEQ ID NO: 3 and amino acids 181-415 of SEQ IDNO: 5). Alternatively, the greatest degree of identity occurs in the SH2domain (i.e., amino acid residues 1-118 of SEQ ID NO:1 and amino acidresidues 50-166 of SEQ ID NO: 5) In a further embodiment, the isolatednucleic acid molecule comprises DNA encoding a Nsp polypeptide havingamino acid residues: (a) 1 to 576 of FIG. 1 (SEQ ID NO: 1), (b) 1 to 501of FIG. 2 (SEQ ID NO: 3), or (c) 1 to 703 of FIG. 3 (SEQ ID NO: 5); oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the invention provides anucleic acid of the full length protein of clones DNA30676-1223 (SEQ IDNO:2), DNA40575-1223 (SEQ ID NO: 4) and DNA61601-1223 (SEQ ID NO:6),deposited with the ATCC under accession number ATCC 209567, ATCC 209565and ATCC 209713, respectively, alternatively the coding sequence ofclones DNA30676-1223, DNA40575-1223 and DNA61601-1223, deposited underaccession number ATCC 209567, ATCC 209565 and ATTC 209713, respectively.

In yet another embodiment, the invention provides a vector comprisingDNA encoding a PRO201, PRO 308, or PRO309 polypeptide. A host cellcomprising such a vector is also provided. By way of example, the hostcells may be CHO cells, E. col, or yeast. A process for producingPRO201, PRO308 or PRO309 polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of PRO201,PRO308 or PRO309 and recovering the same from the cell culture.

In yet another embodiment, the invention provides isolated PRO201,PRO308 or PRO309 polypeptide. In particular, the invention providesisolated native sequence PRO201, PRO308 OR PRO309 polypeptide, which inone embodiment, includes an amino acid sequence comprising residues 1 to576 of FIG. 1 (SEQ ID NO: 1); 1 to 501 of FIG. 2 (SEQ ID NO: 3) or 1 to703 of FIG. 3 (SEQ ID NO: 5). Native PRO201, PRO308 or PRO309polypeptides with or without the initiating methionine are specificallyincluded. Alternatively, the invention provides a PRO201, PRO308 orPRO309 polypeptide encoded by the nucleic acid deposited under accessionnumber ATCC 209567, ATCC 209565 and ATTC209713, respectively.

In yet another embodiment, the invention provides chimeric moleculescomprising a PRO201, PRO308 or PRO309 polypeptide fused to aheterologous polypeptide or amino acid sequence. An example of such achimeric molecule comprises a PRO201, PRO308 OR PRO309 polypeptide fusedto an epitope tag sequence or an Fc region of an immunoglobulin.

In yet another embodiment, the invention provides an antibody whichspecifically binds to PRO201, PRO308 or PRO309 polypeptide. Optionally,the antibody is a monoclonal antibody.

In yet another embodiment, the invention provides for compounds andmethods for developing antagonists against and agonists promoting theNsp1, Nsp2 and/or Nsp3 modulated cellular signaling. In particular, anantagonist of Nsp 1, Nsp2 and/or Nsp3 which blocks, inhibits and/orneutralizes the normal functioning of the latter compounds in cellularsignaling., including both small bioorganic molecules and antisensenucleotides.

In yet another embodiment, the invention provides for alternativelyspliced variants of PRO201, 308 or PRO309.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C shows both the nucleic acid sequence of DNA30676 (SEQ IDNO:2) as well as the encoded amino acid sequence of a native sequencePRO201 polypeptide (SEQ ID NO: 1).

FIGS. 2A-2C shows both the nucleic acid sequence of DNA40575 (SEQ IDNO:4) as well as the encoded amino acid sequence of a native sequencePRO308 polypeptide (SEQ ID NO: 3).

FIGS. 3A-3C show both the nucleic acid sequence of DNA61601 (SEQ IDNO:6)as well as the encoded amino acid sequence of a native sequencePRO309 polypeptide (SEQ ID NO:5).

FIGS. 4A-C shows the sequences of 1328938, 104191 and 1651811 (SEQ IDNOS:13-15), respectively, of the (LIFESEQ™ database, IncytePharmaceuticals, Palo Alto, Calif.), which were used to isolate the fulllength DNA30676, DNA40575 and DNA61601 nucleic acid sequences of theinvention.

FIG. 5A shows the oligonucleotide sequences (SEQ ID NO: 7, SEQ ID NO: 8)which were used in the isolation of DNA30676. FIG. 5B shows theoligonucleotide sequences (SEQ ID NO: 9, SEQ ID NO: 10) which were usedin the isolation of DNA40575. FIG. 5C shows the oligonucleotidesequences (SEQ ID NO: 11, SEQ ID NO: 12) which were used in theisolation of DNA61601.

FIG. 6A shows a figurative illustrative comparison of the variousdomains between a native sequence Nsp1 (SEQ ID NO:1), a native sequenceNsp2 (SEQ ID NO:3) and a native sequence Nsp3 (SEQ ID NO:5);

FIG. 6B show an actual comparison between the 3 sequences themselvesintroducing gaps, as necessary in order to maximize the overall degreeof identity between the three sequences.

FIG. 7 shows the sequence of a native sequence Nsp1 (SEQ ID NO:1)wherein the SH2 regions is identified by overscore and the prolines andserines of the P/S region are indicated in single and double underline,respectively.

FIG. 8 shows a comparison of a native sequence Nsp1 (SEQ ID NO:1) withhuman Shc (SEQ ID NO:16), Sck (SEQ ID NO:17) and Fes (SEQ ID NO:18)proteins.

FIG. 9A is a northern blot showing significant expression of a nativesequence Nsp1 (SEQ ID NO:1) in human fetal liver, while a nativesequence Nsp2 (SEQ ID NO:3) and a native sequence Nsp3 (SEQ ID NO:5)were more widely expressed. FIG. 9B shows the expression of two Nsp3transcripts in hematopoietic tissues.

FIGS. 10A and 10C are western blots wherein anti-flag immunoprecipitateswere blotted with anti-flag, anti-(P)Tyr or anti-Cas antibodies asindicated. In FIG. 10B, anti-EGF receptor (CalBiochem)immunoprecipitates were blotted with anti-flag or anti-(P)Tyr antibodiesas indicated.

FIG. 11 is a western blot showing immunoprecipitates with anti-Flag oranti-p130^(Cas) and blotting with anti-(P)Tyr Ab PY-20 oranti-p130^(Cas).

FIG. 12A is a western blot showing reduced phosphorylation of Nsp1 upontreatment with insulin. FIG. 12B is western blot created by strippingthe blot in FIG. 12A which was reprobed with anti-p130^(Cas) to confirmthat the 130 kD protein was in fact p130^(Cas).

FIG. 13 is a western blot of various Nsp1 mutants which have beentransfected into COS cells and treated with EGF, and the cell lysatesimmunoprecipitated with anti-flag Ab and Western blotted with eitheranti-(P)Tyr, anti-p130^(Cas) or the anti-flag Ab.

FIG. 14A is a micrograph of an ultrathin section of retroviral-infectedvector MSCV NIH3T3 cells, while FIG. 14B show Nsp1 transfected NIH3T3cells. FIG. 14C is a micrograph of an ultrathin section of four weektumors which were fixed, blocked and sections stained with hematoxylinand eosin.

FIG. 15 is a bar graph of tumor size comparing vector controltransfection and Nsp1.sc1, .sc2 and .sc3 cells.

FIG. 16 is a bar graph of showing resistance to apoptosis of gronvthfactor deprived NIH3T3 of transformed subclones Nsp.sub1.1, Nsp₂.1,nontransformed cell culture (NIH3T3-Nsp1.non-trans) and the controlcells NIH3T3-neo.

FIG. 17 is a western blot of COS cells transfected with pRK or Nsp 1,treated with EGF or no treatment, which were lysed in CoIP buffer andincubated with PI3-kinase N-terminal or C-terminal SH2 domain-GST beads(UBI). The precipitated Nsp1 was detected with anti-flag antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

1. Definitions

The terms "PRO201, PRO308 or PRO309," "PRO201, PRO308 or PRO309polypeptide" and "Nsp1, Nsp2 or Nsp3," respectively, when used hereinencompass native sequence PRO201, PRO308 or PRO309, respectively, andPRO201, PRO308 or PRO309 variants, respectively, (which are furtherdefined herein). The may be isolated from a variety of sources, such asfrom human tissue types or from another source, or prepared byrecombinant or synthetic methods.

A "native sequence PRO201, PRO308 or PRO309" comprises a polypeptidehaving the same amino acid sequence as a PRO201, PRO308 or PRO309derived from nature. Such native sequence PRO201, PRO308 or PRO309 canbe isolated from nature or can be produced by recombinant or syntheticmeans. The term "native sequence PRO201, PRO308 or PRO309" specificallyencompasses naturally-occurring truncated or secreted forms of PRO201,PRO308 or PRO309 (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of PRO201, PRO308 or PRO309. Inone embodiment of the invention, the native sequence PRO201, PRO308 orPRO309 is a mature or full-length native sequence PRO201, PRO308 orPRO309 comprising: (a) amino acids 1 to 576 of FIG. 1 (SEQ ID NO: 1);(b) amino acids 1 to 501 of FIG. 2 (SEQ ID NO: 3) and (c) amino acids 1to 703 of FIG. 3 (SEQ ID NO: 5), respectively, with or without theN-terminal signal sequence, and with or without the initiatingmethionine at position 1.

"PRO201, PRO308 or PRO309 variant" means an active PRO201, PRO308 orPRO309 as defined below having at least about 80% amino acid sequenceidentity to: (a) a DNA molecule encoding a PRO201, PRO308 or PRO309polypeptide, with or without its native signal sequence, or (b) thecomplement of the DNA molecule of (a). In a particular embodiment, thePRO201, PRO308 or PRO309 variant has at least about 80% amino acidsequence homology with the PRO201, PRO308 or PRO309 having the deducedamino acid sequence shown in FIG. 1 (SEQ ID NO: 1, FIG. 2 (SEQ ID NO: 3)or FIG. 3 (SEQ ID NO: 5) respectively for a full-length native sequencePRO201, PRO308 or PRO309. Such PRO201, PRO308 or PRO309 variantsinclude, for instance, PRO201, PRO308 or PRO309 polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-termninus of the sequence of FIG. 1 (SEQ ID NO:1), FIG. 2 (SEQ ID NO:3) or FIG. 3 (SEQ ID NO: 5) respectively. Preferably, the nucleic acidor amino acid sequence identity is at least about 85%, more preferablyat least about 90%, and even more preferably at least about 95%.

"Percent (%) amino acid sequence identity" with respect to the PRO201,PRO308 or PRO309 sequences identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the PRO201, PRO308 or PRO309sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST-2 software set to the default parameters. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared.

"Percent (%) nucleic acid sequence identity" with respect to the PRO201,PRO308 or PRO309 sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO201, PRO308 or PRO309 sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST-2 software set thedefault parameters. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared.

"Isolated," when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO201, PRO308 orPRO309 natural environment will not be present. Ordinarily, however,isolated polypeptide will be prepared by at least one purification step.

An "isolated" DNA30676 (SEQ ID NO:2), DNA40575 (SEQ ID NO:4) or DNA61601(SEQ ID NO:6) nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe DNA30676, DNA40575 or DNA61601 nucleic acid. An isolated DNA30676SEQ ID NO:2, DNA40575 or DNA61601 nucleic acid molecule is other than inthe form or setting in which it is found in nature. Isolated DNA30676,DNA40575 or DNA61601 nucleic acid molecules therefore are distinguishedfrom the DNA30676, DNA40575 or DNA61601 nucleic acid molecule as itexists in natural cells. However, an isolated DNA30676, DNA40575 orDNA61601 nucleic acid molecule includes DNA30676, DNA40575 or DNA61601nucleic acid molecules contained in cells that ordinarily expressDNA30676, DNA40575 or DNA61601 where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term "control sequences" refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, "operably linked"means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term "antibody" is used in the broadest sense and specificallycovers single anti-PRO201, anti-PRO308 or anti-PRO309 monoclonalantibodies (including agonist, antagonist, and neutralizing antibodies)and anti-PRO201, anti-PRO308 or anti-PRO309 antibody compositions withpolyepitopic specificity. The term "monoclonal antibody" as used hereinrefers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts.

"Active" or "activity" for the purposes herein refers to form(s) ofPRO201, PRO308 or PRO309 which retain the biologic and/or immunologicactivities of native or naturally-occurring PRO201, PRO308 or PRO309. Apreferred activity is the ability to bind to and affect, e.g., block orotherwise modulate, an activity of cellular response to externalsignaling. The activity preferably involves the regulation oftumorigenesis and response to stimulation by integrin receptors and byepidermal growth factor (EGF), insulin growth factor (IGF) and throughother receptor tyrosine (RTK) ligands.

The term "modulate" means to affect (eg., either upregulate,downregulate or otherwise control) the level of a signaling pathway.Cellular processes under the control of signal transduction include, butare not limited to, transcription of specific genes, normal cellularfunctions, such as metabolism, proliferation, differentiation, adhesion,apoptosis and survival, as well as abnormal processes, such astransformation, blocking of differentiation and metastasis.

The term "antagonist" is used herein in the broadest sense to includeany molecule which blocks, prevents, inhibits, or neutralizes theprocess by which the Nsp1, Nsp2 and Nsp3 molecules of the invention thatinterferes with the interaction of any of the protein domains of Nsp1,Nsp2 and Nsp3 with various target proteins. Such interactions cangenerally occur with the C-terminal end of Nsp1, Nsp2, Nsp3, orspecifically the SH2 and/or proline/serine (P/S) rich regions withphosphotyrosyl residues and polyproline motifs with a target bindingsite. In a similar manner, the term "agonist" is used herein to includeany molecule which promotes, enhances or stimulates the interaction ofthe protein domains of Nsp1, Nsp2 and Nsp3 including the SH2 and/orproline/serine (P/S) rich regions with phosphotyrosyl residues andpolyproline motifs, respectively on various target proteins. Suitablemolecules that affect the interaction of the SH2 and/or P/S regions ofNsp1, Nsp2, Nsp3 and the phosphotyrosyl residues and polyproline motifs,respectively or target proteins include fragments of the latter or smallbioorganic molecules, e.g., peptidomimetics, which will prevent orenhance, as the case may be, the interaction. Non-limiting examplesinclude proteins, peptides, glycoproteins, glycopeptides, glycolipids,polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules,peptidomimetics, pharmacological agents and their metabolites,transcriptional and translation control sequences, and the like. Anotherpreferred form of antagonist includes antisense nucleotides that inhibitthe Nsp1, Nsp2 or Nsp3 modulated signaling. Preferred forms bind tospecific regions on either Nsp1, Nsp2 or Nsp3 or the targets with whichNsp 1, Nsp2 or Nsp3 interact.

II. Compositions and Methods of the Invention

A. Full-length Nsp1, Nsp2 or Nsp3

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO201 (Nsp1), PRO308 (Nsp2) or PRO309 (Nsp3). In particular,Applicants have identified and isolated cDNA encoding a PRO201, PRO308and PRO309 polypeptide, as disclosed in further detail in the Examplesbelow. Using the BLAST-2 sequence alignment computer program set to thedefault parameters, Applicants found that a full-length native sequencePRO201 and PRO309 (shown in FIGS. 6A & 6B and SEQ ID NO: 1-6) haveregions of SH2 homology and PRO201, PRO308 and PRO309 have aproline/serine rich (P/S) region homology. SH2 domains are known to bindspecific phosphotyrosyl residues, while the P/S region could be apotential SH3 interaction domain. Accordingly, it is presently believedthat PRO201, PRO308 and PRO309 disclosed in the present application arenewly identified family of adaptor proteins and may possess propertieswhich modulate intracellular signaling pathways.

B. PRO201, PRO308 or PRO309 Variants

In addition to the full-length native sequence PRO201, PRO308 and PRO309described herein, it is contemplated that PRO201, PRO308 and PRO309variants can be prepared. PRO201, PRO308 and PRO309 variants can beprepared by introducing appropriate nucleotide changes into the PRO201,PRO308 or PRO309 DNA, or by synthesis of the desired PRO201, PRO308 orPRO309 polypeptides. Those skilled in the art will appreciate that aminoacid changes may alter post-translational processes of the PRO201,PRO308 or PRO309, such as changing the number or position ofglycosylation sites or altering the membrane anchoring characteristics.

Variations in the native full-length sequence PRO201, PRO308 or PRO309or in various domains of the PRO201, PRO308 or PRO309 described herein,can be made, for example, using any of the techniques and guidelines forconservative and non-conservative mutations set forth, for instance, inU.S. Pat. No. 5,364,934. Variations may be a substitution, deletion orinsertion of one or more codons encoding the PRO201, PRO308 or PRO309that results in a change in the amino acid sequence of the PRO201,PRO308 or PRO309 as compared with the native sequence PRO201, PRO308 orPRO309. Optionally the variation is by substitution of at least oneamino acid with any other amino acid in one or more of the domains ofthe PRO201, PRO308 or PRO309. Guidance in determining which amino acidresidue may be inserted, substituted or deleted without adverselyaffecting the desired activity may be found by comparing the sequence ofthe PRO201, PRO308 or PRO309 with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of 1 to 5 aminoacids. The variation allowed may be determined by systematically makinginsertions, deletions or substitutions of amino acids in the sequenceand testing the resulting variants for activity in the in vitro assaydescribed in the Examples below.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA. 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO201, PRO308 or PRO309 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Alanine is alsotypically preferred because it is the most common amino acid. Further,it is frequently found in both buried and exposed positions [Creighton,The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol. 150:1(1976)]. If alanine substitution does not yield adequate amounts ofvariant, an isoteric amino acid can be used.

C. Modifications of PRO201. PRO308 OR PRO309

Covalent modifications of PRO201, PRO308 or PRO309 are included withinthe scope of this invention. One type of covalent modification includesreacting targeted amino acid residues of the PRO201, PRO308 or PRO309with an organic derivatizing agent that is capable of reacting withselected side chains or the N- or C-terminal residues of the PRO201,PRO308 or PRO309. Derivatization with bifunctional agents is useful, forinstance, for crosslinking PRO201, PRO308 or PRO309 to a water-insolublesupport matrix or surface for use in the method for purifyinganti-PRO201, PRO308 or PRO309 antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of Iysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO201, PRO308 or PRO309polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. "Alteringthe native glycosylation pattern" is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequencePRO201, PRO308 or PRO309, and/or adding one or more glycosylation sitesthat are not present in the native sequence PRO201, PRO308 or PRO309,and/or alteration of the ratio and/or composition of the sugar residuesattached to the glycosylation site(s).

Addition of glycosylation sites to the PRO201, PRO308 or PRO309polypeptide may be accomplished by altering the amino acid sequence. Thealteration may be made, for example, by the addition of, or substitutionby, one or more serine or threonine residues to the native sequencePRO201, PRO308 or PRO309 (for O-linked glycosylation sites). The PRO201,PRO308 or PRO309 amino acid sequence may optionally be altered throughchanges at the DNA level, particularly by mutating the DNA encoding thePRO201, PRO308 or PRO309 polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO201, PRO308 or PRO309 polypeptide is by chemical or enzymaticcoupling of glycosides to the polypeptide. Such methods are described inthe art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO201, PRO308 or PRO309polypeptide may be accomplished chemically or enzymatically or bymutational substitution of codons encoding for amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of PRO201, PRO308 or PRO309comprises linking the PRO201, PRO308 or PRO309 polypeptide to one of avariety of nonproteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

The PRO201, PRO308 or PRO309 of the present invention may also bemodified in a way to form a chimeric molecule comprising PRO201, PRO308or PRO309 fused to another, heterologous polypeptide or amino acidsequence. In one embodiment, such a chimeric molecule comprises a fusionof the PRO201, PRO308 or PRO309 with a tag polypeptide which provides anepitope to which an anti-tag antibody can selectively bind. The epitopetag is generally placed at the amino- or carboxyl- terminus of thePRO201, PRO308 or PRO309. The presence of such epitope-tagged forms ofthe PRO201, PRO308 or PRO309 can be detected using an antibody againstthe tag polypeptide. Also, provision of the epitope tag enables thePRO201, PRO308 or PRO309 to be readily purified by affinity purificationusing an anti-tag antibody or another type of affinity matrix that bindsto the epitope tag. In an alternative embodiment, the chimeric moleculemay comprise a fusion of the PRO201, PRO308 or PRO309 with animmunoglobulin or a particular region of an immunoglobulin. For abivalent form of the chimeric molecule, such a fusion could be to the Fcregion of an IgG molecule.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

D. Preparation of PRO201 PRO308 OR PRO309

The description below relates primarily to production of PRO201, PRO308OR PRO309 by culturing cell transformed or transfected with a vectorcontaining PRO201, PRO308 OR PRO309 nucleic acid. It is, of course,contemplated that alternative methods, which are well known in the art,may be employed to prepare PRO201, PRO308 or PRO309. For instance, thePRO201, PRO308 or PRO309 sequence, or portions thereof, may be producedby direct peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO201, PRO308 or PRO309 may be chemically synthesized separately andcombined using chemical or enzymatic methods to produce the full-lengthPRO201, PRO308 or PRO309.

1. Isolation of DNA Encoding PRO201, PRO308 or PRO309

DNA encoding PRO201, PRO308 or PRO309 may be obtained from a cDNAlibrary prepared from tissue believed to possess the PRO201, PRO308 orPRO309 mRNA and to express it at a detectable level. Accordingly, humanPRO201, PRO308 or PRO309 DNA can be conveniently obtained from a cDNAlibrary prepared from human tissue, such as described in the Examples.The PRO201, PRO308 or PRO309-encoding gene may also be obtained from agenomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the PRO201,PRO308 or PRO309 or oligonucleotides of at least about 20-80 bases)designed to identify the gene of interest or the protein encoded by it.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures, such as described in Sambrook etal., Molecular Cloning: A Laboratorv Manual (New York: Cold SpringHarbor Laboratory Press, 1989). An alternative means to isolate the geneencoding PRO201, PRO308 or PRO309 is to use PCR methodology [Sambrook etal., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (ColdSpring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³² P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using computer software programssuch as BLAST, BLAST-2, ALIGN, DNAstar, and INHERIT which employ variousalgorithms to measure homology.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO201, PRO308 OR PRO309 production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH and the like, can be selected by the skilled artisanwithout undue experimentation. In general, principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: a Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, CaPO4 and electroporation. Depending on the host cell used,transformation is performed using standard techniques appropriate tosuch cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., supra, or electroporation is generallyused for prokaryotes or other cells that contain substantial cell-wallbarriers. Infection with Agrobacterizim tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact. 130:946 (1977) and Hsiao et al.,Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods forintroducing DNA into cells, such as by nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orpolycations, e.g., polybrene, polyornithine, may also be used. Forvarious techniques for transforming mammalian cells, see Keown et al.,Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature,336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for PRO201,PRO308 OR PRO309-encoding vectors. Saccharomnyces cerevisiae is acommonly used lower eukaryotic host microorganism.

Suitable host cells for the expression of glycosylated PRO201, PRO308 ORPRO309 are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells. Examples of useful mammalianhost cell lines include Chinese hamster ovary (CHO) and COS cells. Morespecific examples include monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlauband Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertolicells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mousemammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriatehost cell is deemed to be within the skill in the art.

3. Selection and Use of a Renlicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO201, PRO308 ORPRO309 may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

The PRO201, PRO308 or PRO309 may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the PRO201, PRO308 or PRO309 DNA that isinserted into the vector. The signal sequence may be a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2: plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, eg., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the PRO201,PRO308 or PRO309 nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trpl gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 2:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO201, PRO308 or PRO309 nucleic acid sequence to directmRNA synthesis. Promoters recognized by a variety of potential hostcells are well known. Promoters suitable for use with prokaryotic hostsinclude the β-lactamase and lactose promoter systems [Chang et al.,Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingPRO201, PRO308 or PRO309.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO201, PRO308 or PRO309 transcription from vectors in mammalian hostcells is controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, and from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding the PRO201, PRO308 or PRO309 by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5' or 3' to thePRO201, PRO308 or PRO309 coding sequence, but is preferably located at asite 5' from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5' and, occasionally 3', untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO201, PRO308 or PRO309.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO201, PRO308 or PRO309 in recombinant vertebrate cellculture are described in Gething et al., Nature 293:620-625 (1981);Mantei et al., Nature, 281:4046 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO201, PRO308 or PRO309 polypeptide or against a synthetic peptidebased on the DNA sequences provided herein or against exogenous sequencefused to PRO201, PRO308 or PRO309 DNA and encoding a specific antibodyepitope.

5. Purification of Polypeptide

Forms of PRO201, PRO308 or PRO309 may be recovered from culture mediumor from host cell lysates. If membrane-bound, it can be released fromthe membrane using a suitable detergent solution (eg. Triton-X 100) orby enymatic cleavage. Cells employed in expression of PRO201, PRO308 orPRO309 can be disrupted by various physical or chemical means, such asfreeze-thaw cycling, sonication, mechanical disruption, or cell lysingagents.

It may be desired to purify PRO201, PRO308 or PRO309 from recombinantcell proteins or polypeptides. The following procedures are exemplary ofsuitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; protein A Sepharose columns to removecontaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of the PRO201, PRO308 or PRO309. Various methods ofprotein purification may be employed and such methods are known in theart and described for example in Deutscher, Methods in Enzymology, 182(1990); Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York (1982). The purification step(s) selected willdepend, for example, on the nature of the production process used andthe particular PRO201, PRO308 or PRO309 produced.

E. Uses for PRO201, PRO308 OR PRO309

Nucleotide sequences (or their complement) encoding PRO201, PRO308 orPRO309 have various applications in the art of molecular biology,including uses as hybridization probes, in chromosome and gene mappingand in the generation of anti-sense RNA and DNA. PRO201, PRO308 orPRO309 nucleic acid will also be useful for the preparation of PRO201,PRO308 or PRO309 polypeptides by the recombinant techniques describedherein.

The full-length native sequence PRO201, PRO308 or PRO309 (SEQ ID NO:1, 3& 5) gene, or portions thereof, may be used as hybridization probes fora cDNA library to isolate the full-length gene or to isolate still othergenes (for instance, those encoding naturally-occurring variants ofPRO201, PRO308 or PRO309 or PRO201, PRO308 or PRO309 from other species)which have a desired sequence identity to the PRO201, PRO308 or PRO309sequence disclosed in FIG. 1-3 (SEQ ID NO: 1, 3 & 5). Optionally, thelength of the probes will be about 20 to about 50 bases. Thehybridization probes may be derived from the nucleotide sequence of SEQID NO: 1, 3 or 5 or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO201, PRO308 or PRO309. By wayof example, a screening method will comprise isolating the coding regionof the PRO201, PRO308 or PRO309 gene using the known DNA sequence tosynthesize a selected probe of about 40 bases. Hybridization probes maybe labeled by a variety of labels, including radionucleotides such as ³²P or ³⁵ S, or enzymatic labels such as alkaline phosphatase coupled tothe probe via avidintbiotin coupling systems. Labeled probes having asequence complementary to that of the PRO201, PRO308 or PRO309 gene ofthe present invention can be used to screen libraries of human cDNA,genomic DNA or mRNA to determine which members of such libraries theprobe hybridizes to. Hybridization techniques are described in furtherdetail in the Examples below.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO201, PRO308 or PRO309sequences.

Nucleotide sequences encoding a PRO201, PRO308 or PRO309 can also beused to construct hybridization probes for mapping the gene whichencodes that PRO201, PRO308 or PRO309 and for the genetic analysis ofindividuals with genetic disorders. The nucleotide sequences providedherein may be mapped to a chromosome and specific regions of achromosome using known techniques, such as in situ hybridization,linkage analysis against known chromosomal markers, and hybridizationscreening with libraries.

When the coding sequences for PRO201, PRO308 or PRO309 encode a proteinwhich binds to another protein (example, where the PRO201, PRO308 orPRO309 is a receptor), the PRO201, PRO308 or PRO309 can be used inassays to identify the other proteins or molecules involved in thebinding interaction. By such methods, inhibitors of the receptor/ligandbinding interaction can be identified. Proteins involved in such bindinginteractions can also be used to screen for peptide or small moleculeinhibitors or agonists of the binding interaction. Also, the receptorPRO201, PRO308 or PRO309 can be used to isolate correlative ligand(s).Screening assays can be designed to find lead compounds that mimic thebiological activity of a native PRO201, PRO308 or PR0309 or a receptorfor PRO201, PRO308 or PRO309. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart.

Nucleic acids which encode PRO201, PRO308 or PRO09 or its modified formscan also be used to generate either transgenic animals or "knock out"animals which, in turn, are useful in the development and screening oftherapeutically useful reagents. A transgenic animal (e.g., a mouse orrat) is an animal having cells that contain a transgene, which transgenewas introduced into the animal or an ancestor of the animal at aprenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, cDNA encoding PRO201, PRO308 or PRO309 canbe used to clone genomic DNA encoding PRO201, PRO308 or PRO309 inaccordance with established techniques and the genomic sequences used togenerate transgenic animals that contain cells which express DNAencoding PRO201, PRO308 or PRO309. Methods for generating transgenicanimals, particularly animals such as mice or rats, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009. Typically, particular cells would betargeted for PRO201, PRO308 or PRO309 transgene incorporation withtissue-specific enhancers. Transgenic animals that include a copy of atransgene encoding PRO201, PRO308 or PRO309 introduced into the germline of the animal at an embryonic stage can be used to examine theeffect of increased expression of DNA encoding PRO201, PRO308 or PRO309.Such animals can be used as tester animals for reagents thought toconfer protection from, for example, pathological conditions associatedwith its overexpression. In accordance with this facet of the invention,an animal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

Non-human homologues of PRO201, PRO308 or PRO309 can be used toconstruct a PRO201, PRO308 or PRO309 "knock out" animal which has adefective or altered gene encoding PRO201, PRO308 or PRO309 as a resultof homologous recombination between the endogenous gene encoding PRO201,PRO308 or PRO309 and altered genomic DNA encoding PRO201, PRO308 orPRO309 introduced into an embryonic cell of the animal. For example,cDNA encoding PRO201, PRO308 or PRO309 can be used to clone genomic DNAencoding PRO201, PRO308 or PRO309 in accordance with establishedtechniques. A portion of the genomic DNA encoding PRO201, PRO308 orPRO309 can be deleted or replaced with another gene, such as a geneencoding a selectable marker which can be used to monitor integration.Typically, several kilobases of unaltered flanking DNA (both at the 5'and 3' ends) are included in the vector [see e.g., Thomas and Capecchi,Cell, 51:503 (1987) for a description of homologous recombinationvectors]. The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced DNA hashomologously recombined with the endogenous DNA are selected [see e.g.,Li et al., Cell, 69:915 (1992)]. The selected cells are then injectedinto a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a "knock out" animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the PRO201, PRO308 or PRO309 polypeptide.

DNA30676 contains a single long open reading frame which can encode a576 amino acid protein, herein termed Nsp1 (novel SH2 containingprotein) (FIG. 1, SEQ ID NO: 2). Nsp1 is related to Sck and Shc (SeeFIG. 8) as determined by the indicated N-terminal homology. Nsp1 alsocontains a proline-serine rich domain (P/S) in the middle of the proteinthat may function as an SH3 interaction domain. The C terminus of theprotein has no obviously relevant homology to any known mammalianproteins. Nsp1, Nsp2 and Nsp3 share an overall homology between 33 and47%. Nsp3 has an SH2 domain and a potential SH3 interaction domain,while Nsp2 lacks the SH2 domain but does have a potential SH3interaction domain. The absence of the SH2 domain in Nsp2 is suggestivethat this protein could act as a dominant negative regulator of theother two Nsps. The lack of any apparent kinase or phosphatase domainsuggests that they represent a novel family of adaptor proteins.

Adapter proteins are believed to play a significant role integratingmultiple signaling cascades and in determining specific response toextracellular stimuli. Signals generated by growth factors such as EGFor IGF-1 through receptor tyrosine kinases or by extracellular matrixcomponents acting through the integrin receptors can induce cytoskeletalchanges. Applicants have shown that the EGF receptorcoimmunoprecipitates with Nsp1 and is phosphorylated in response to EGFsignaling. Thus, antagonists of Nsp1 would be expected to be useful toinhibit cellular response attributed to stimulation by growth factorssuch as EGF or IGF-1 (e.g, tumorigenesis).

Several characteristics suggest that Nsp1 could play an important rolein modulating the response to external stimuli. Nsp1 is phosphorylatedin response to EGF stimulation and forms a complex that includes the EGFreceptor, PI3 kinase and Cas. The Nsp1/Cas complex also responds tosignaling through the fibronectin receptor. However, the stoichiometryof the interaction and the phosphorylation status of the componentsdiffers between the two stimuli. The implication is that that biologicaloutcome in response to these extracellular signals could be quitedistinct in the presence or absence of Nsp1. For example, FAK associateswith the SH3 region of Cas via a PXXP region at the C-terminus of FAK(P(715)SRP - mouse nomenclature (Harte et al., J. Biol. Chem. 271:13649-55 (1996). There are six PXXP signatures in Nsp1. This raises thepossibility that Nsp1 could compete for the SH3 region on Cas anddecrease the amount of Fak that is bound to Cas and so alter Fakdependent events. The data also point to an EGF mediated decrease in theextent of phosphorylation of the Cas that is associated with Nsp1. Thiscomplex then is likely to have a decrease in the number of proteinsassociated with the phosphorylated tyrosines of the Cas and so lead tochanges in downstream events. As Nsp1 expression is highest in fetaltissues this protein could potentially have an important role inmediating the developmental readout of extracellular signals.

F. Anti-PRO201, PRO308 or PRO309 Antibodies

The present invention further provides anti-PRO201, anti-PRO308 oranti-PRO309 antibodies. Exemplary antibodies include polyclonal,monoclonal, humanized, bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-PRO201, anti-PRO308 or anti-PRO309 antibodies may comprisepolyclonal antibodies. Methods of preparing polyclonal antibodies areknown to the skilled artisan. Polyclonal antibodies can be raised in amammal, for example, by one or more injections of an immunizing agentand, if desired, an adjuvant. Typically, the immunizing agent and/oradjuvant will be injected in the mammal by multiple subcutaneous orintraperitoneal injections. The immunizing agent may include the PRO201,PRO308 or PRO309 polypeptide or a fusion protein thereof. It may beuseful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins include but are not limited to keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examplesof adjuvants which may be employed include Freund's complete adjuvantand MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalosedicorynomycolate). The immunization protocol may be selected by oneskilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-PRO201, anti-PRO308 or anti-PRO309 antibodies may,alternatively, be monoclonal antibodies. Monoclonal antibodies may beprepared using hybridoma methods, such as those described by Kohler andMilstein, Nature, 256:495 (1975). In a hybridoma method, a mouse,hamster, or other appropriate host animal, is typically immunized withan immunizing agent to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the immunizingagent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the PRO201, PRO308 or PRO309polypeptide or a fusion protein thereof. Generally, either peripheralblood lymphocytes ("PBLs") are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine ("HAT medium"), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed againstPRO201, PRO308 or PRO309. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to preventcrosslinkinga.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Humanized Antibodies

The anti-PRO201, PRO308 or PRO309 antibodies of the invention mayfurther comprise humanized antibodies or human antibodies. Humanizedforms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as "import" residues, whichare typically taken from an "import" variable domain. Humanization canbe essentially performed following the method of Winter and coworkers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such "humanized" antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)].

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe PRO201, PRO308 or PRO309, the other one is for any other antigen,and preferably for a cell-surface protein or receptor or receptorsubunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Trauneckeret al., EMBO J.,10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

G. Uses for anti-PRO201, PRO308 or PRO309 Antibodies

The anti-PRO201, anti-PRO308 or anti-PRO309 antibodies of the inventionhave various utilities. For example, anti-PRO201, anti-PRO308 oranti-PRO309 antibodies may be used in diagnostic assays for PRO201,PRO308 or PRO309, eg., detecting its expression in specific cells,tissues, or serum. Various diagnostic assay techniques known in the artmay be used, such as competitive binding assays, direct or indirectsandwich assays and immunoprecipitation assays conducted in eitherheterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: AManual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. Theantibodies used in the diagnostic assays can be labeled with adetectable moiety. The detectable moiety should be capable of producing,either directly or indirectly, a detectable signal. For example, thedetectable moiety may be a radioisotope, such as ³ H, ¹⁴ C, ³² P, ³⁵ S,or ¹²⁵ I, a fluorescent or chemiluminescent compound, such asfluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, suchas alkaline phosphatase, beta-galactosidase or horseradish peroxidase.Any method known in the art for conjugating the antibody to thedetectable moiety may be employed, including those methods described byHunter et al., Nature, 144:945 (1962); David et al., Biochemistry,13:1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); andNygren, J. Histochem. and Cytochem., 30:407 (1982).

Anti-PRO201, anti-PR0308 or anti-PRO309 antibodies also are useful forthe affinity purification of PRO201, PRO308 or PRO309 from recombinantcell culture or natural sources. In this process, the antibodies againstPRO201, PRO308 or PRO309 are immobilized on a suitable support, such aSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing thePRO201, PRO308 or PRO309 to be purified, and thereafter the support iswashed with a suitable solvent that will remove substantially all thematerial in the sample except the PRO201, PRO308 or PRO309, which isbound to the immobilized antibody. Finally, the support is washed withanother suitable solvent that will release the PRO201, PRO308 or PRO309from the antibody.

Anti-PRO201, anti-PRO308 or anti-PRO309 would also have similarutilities to those articulated under uses for PRO201, PRO308 and PRO309polypeptides.

H. PRO201, PRO308 and PRO309 Antagonists

Several approaches may be suitably employed to create the antagonist andagonist compounds of the present invention. Any approach where theantagonist molecule can be targeted to the interior of the cell, whichinterferes or prevents wild type PRO201, PRO308 or PRO309 from normaloperation is suitable. Additional properties of such antagonist oragonist molecules are readily determninable by one of ordinary skill,such as size, charge and hydrophobicity suitable for transmembranetransport.

Where mimics or other mammalian homologues of PRO201, PRO308 or PRO309are to be identified or evaluated, the cells are exposed to the testcompound and compared to positive controls which are exposed only toPRO201, PRO308 or PRO309 and to negative controls which were not exposedto either the compound or the natural ligand. Where antagonists oragonists of PRO201, PRO308 or PRO309 signal modulation are to beidentified or evaluated, the cells are exposed to the compound of theinvention in the presence of the natural ligand and compared to controlswhich are not exposed to the test compound.

Detection assays may by employed as a primary screen to evaluate thephosphatase inhibition/enhancing activity of the antagonist/agonistcompounds of the invention. The assays may also be used to assess therelative potency of a compound by testing a range of concentrations, ina range from 100 mM to 1 pM, for example, and computing theconcentration at which the amount of phosphorylation or signaltransduction is reduced or increased by 50% (IC₅₀) compared to controls.

Assays can be performed to identify compounds that affectphosphorylation of PRO201, PRO308 or PRO309 substrates. Specifically,assays can be performed to identify compounds that increase thephosphorylation activity of PRO201, PRO308 or PRO309 or assays can beperformed to identify compounds that decrease the phosphorylation ofPRO201, PRO308 or PRO309 substrates. These assays can be performedeither on whole cells themselves or on cell extracts. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays, cell based assays,etc. Such assay formats are well known in the art.

The screening assays of the present invention are amenable tohigh-throughput screening of chemical libraries, and are particularlysuitable for identifying small molecule drug candidates.

(1) Antagonist and agonist molecules

To screen for antagonists and/or agonists of PRO201, PRO308 or PRO309signaling, the assay mixture is incubated under conditions whereby, butfor the presence of the candidate pharmacological agent, PRO201, PRO308or PRO309 induces intracellular signaling (for example, association ofNsp1 with the EGF receptor) with a reference activity. The mixturecomponents can be added in any order that provides for the requisiteactivity. Incubation may be performed at any temperature thatfacilitates optimal binding, typically between about 4° and 40° C., morecommonly between about 15° and 40° C. Incubation periods are likewiseselected for optimal binding but also minimized to facilitate rapid,high-throughput screening, and are typically between about 0.1 and 10hours, preferably less than 5 hours, more preferably less than 2 hours.After incubation, the effect of the candidate pharmacological agent onthe PRO201, PRO308 or PRO309 signaling is determined in any convenientway. For cell-free binding-type assays, a separation step is often usedto separate bound and unbound components. Separation may, for example,be effected by precipitation (e.g. TCA precipitation,immunoprecipitation, etc.), immobilization (e.g. on a solid substrate),followed by washing. The bound protein is conveniently detected bytaking advantage of a detectable label attached to it, e.g. by measuringradioactive emission, optical or electron density, or by indirectdetection using, e.g. antibody conjugates.

Suitable molecules that affect the protein-protein interaction ofPRO201, PRO308 or PRO309 and its binding proteins include fragments ofthe latter or small molecules, e.g., peptidomimetics, which will preventinteraction and proper complex formation. Such small molecules, whichare usually less than 10 K molecular weight, are preferable astherapeutics since they are more likely to be permeable to cells, areless susceptible to degradation by various cellular mechanisms, and arenot as apt to elicit an immune response as proteins. Small moleculesinclude but are not limited to synthetic organic or inorganic compounds.Many pharmaceutical companies have extensive libraries of suchmolecules, which can be conveniently screened by using the assays of thepresent invention. Non-limiting examples include proteins, peptides,glycoproteins, glycopeptides, glycolipids, polysaccharides,oligosacchardies, nucleic acids, bioorganic molecules, peptidomimetics,pharmacological agents and their metabolites, transcriptional andtranslation control sequences, and the like.

A preferred technique for identifying molecules which bind to PRO201,PRO308 or PRO309 utilizes a chimeric substrate (e.g., epitope-taggedfused or fused immunoadhesin) attached to a solid phase, such as thewell of an assay plate. The binding of the candidate molecules, whichare optionally labeled (e.g., radiolabeled), to the immobilized receptorcan be measured. Alternatively, competition for interaction of Nsp1 withthe binding proteins can be assayed. Further yet, molecules may bescreened which affect the tumorigenicity of PRO201, PRO308 or PRO309 inNIH3T3 cells in nude mice. In screening for antagonists and/or agonists,PRO201, PRO308 or PRO309 can be exposed to a PRO201, PRO308 or PRO309substrate followed by the putative antagonist and/or agonist, or thePRO201, PRO308 or PRO309 binding protein and antagonist and/or agonistcan be added simultaneously, and the ability of the antagonist and/oragonist to block PRO201, PRO308 or PRO309 activation can be evaluated.

(2) Detection assays

The PRO201, PRO308 or PRO309 polypeptides are useful in assays foridentifying lead compounds for therapeutically active agents thatmodulate PRO201, PRO308 or PRO309 signaling. Specifically, leadcompounds that either prevent the formation of PRO201, PRO308 or PRO309signaling complexes or prevent or attenuate PRO201, PRO308 or PRO309modulated can be conveniently identified.

(a) Biochemical detection techniques

Biochemical analysis techniques can be evaluated by a variety oftechniques. One typical assay mixture which can be used with the presentinvention contains PRO201, PRO308 or PRO309 and a protein with whichPRO201, PRO308 or PRO309 is normally directly or indirectly associated(e.g. Cas), usually in an isolated, partially pure or pure form. One orboth of these components may be PRO201, PRO308 or PRO309 bound toanother peptide or polypeptide, which may, for example, provide orenhance protein-protein binding, improve stability under assayconditions, etc. In addition, one of the components usually comprises oris coupled to a detectable label. The label may provide for directdetection by measuring radioactivity, luminescence, optical or electrondensity, etc., or indirect detection such as an epitope tag, an enzyme,etc. The assay mixture can additionally comprise a candidatepharmacological agent, and optionally a variety of other components,such as salts, buffers, carrier proteins, e.g. albumin, detergents,protease inhibitors, nuclease inhibitors, antimicrobial agents, etc.,which facilitate binding, increase stability, reduce non-specific orbackground interactions, or othervise improve the efficiency orsensitivity of the assay.

The following detection methods may also be used in a cell-free systemwherein cell lysate containing the signal transducing substrate moleculeand PRO201, PRO308 or PRO309 is mixed with a compound of the invention.

(i) Whole cell detection

A common technique involves incubating cells with vertebrate PRO201,PRO308 or PRO309 and radiolabeled phosphate, lysing the cells,separating cellular protein components of the lysate using anSDS-polyacrylamide gel (SDS-PAGE) technique, in either one or twodimensions, and detecting the presence of phosphorylated proteins byexposing X-ray film. Detection can also be effected without usingradioactive labeling. In such a technique, the protein components (e.g.,separated by SDS-PAGE) are transferred to a nitrocellulose membranewhere the presence of phosphorylated tyrosine is detected using anantiphosphotyrosine antibody (anti-pTyr).

Alternatively, the anti-pTyr can be conjugated with an enzyme, such ashorseradish peroxidase, and detected by subsequent addition of acolorimetric substrate for the enzyme. A further alternative involvesdetecting the anti-pTyr by reacting with a second antibody thatrecognizes the anti-pTyr, this second antibody being labeled with eithera radioactive moiety or an enzyme as previously described. Examples ofthese and similar techniques are described in Hansen et al.,Electrophoresis 14: 112-126 (1993); Campbell et al., J. Biol. Chem. 268:7427-7434 (1993); Donato et al., Cell Growth Diff. 3: 258-268 (1992);Katagiri et al., J. Immunol. 150: 585-593 (1993). Additionally, theanti-pTyr can be detected by labeling it with a radioactive substance,followed by scanning the labeled nitrocellulose to detect radioactivityor exposure of X-ray film.

(b) Biological detection techniques:

The ability of the antagonistlagonist compounds of the invention tomodulate the activity PRO201, PRO308 or PRO309, which itself modulatesintracellular signaling, may also be measured by scoring formorphological or functional changes associated with ligand binding. Anyqualitative or quantitative technique known in the art may be appliedfor observing and measuring cellular processes which comes under thecontrol of PRO201, PRO308 or PRO309. For example, expression of nativesequence Nsp1 (SEQ ID NO:2) in NIH3T. cells causes morphologicallytransformation of the cells. The presence and or number of these focican be used as an indicator of biological efficacy of antagonists ofagonists of Nsp1 signaling.

The data obtained from these cell culture assays and animal studies canbe used in formulating a range of dosages for use in humans. The dosageof the compounds of the invention should lie within a range ofcirculating concentrations with little or no toxicity. The dosage mayvary within this range depending on the dosage form employed and theroute of administration.

(2) Antisense nucleotides

Another preferred class of antagonists involves the use of gene therapytechniques, include the administration of antisense nucleotides.Applicable gene therapy techniques include single or multipleadministrations of therapeutically effective DNA or mRNA. Antisense RNAsand DNAs can be used as therapeutic agents for blocking the expressionof certain genes in vivo. Short antisense oligonucleotides can beimported into cells where they act as inhibitors, despite their lowintracellular concentrations caused by restricted uptake by the cellmembrane, Zamecnik et al., Proc. Natl. Acad. Sci. USA 83: 4143-4146(1986). The oligonucleotides can be modified to enhance their uptake,e.g., by substituting their negatively charged phophodiester groups byuncharged groups.

There are a variety of techniques known for introducing nucleic acidsinto viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, ex vivo, or invivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc. Thecurrently preferred in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection, Dzau et al., Trends Biotech. 11:205-210 (1993). In some situations it is desirable to provide thenucleic acid source with an agent that targets the target cells, such asan antibody specific for a cell surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake, e.g.,capsid proteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling, andproteins that target intracellular localization and enhanceintracellular half-life. The technique of receptor-mediated endocytosisis described, for example, by Wu et al., J. Biol. Chem. 262: 4429-4432(1987); Wagner et al., Proc. Natl. Acad. Sci. USA 87: 3410-3414 (1990).For a review of known gene marking and gene therapy protocols, seeAnderson et al., Science 256: 808-813 (1992).

In one embodiment, PRO201, PRO308 or PRO309 antagonist and/or agonistmolecules may be used to bind endogenous ligand in the cell, therebycausing the cell to be unresponsive to PRO201, PRO308 or PRO309 wildtype, especially when the levels of PRO201, PRO308 or PRO309 in the cellexceed normal physiological levels. Also, it may be beneficial to bindendogenous PRO201, PRO308 or PRO309 substrates or complexing agents thatare activating undesired cellular responses (such as proliferation oftumor cells).

In a further embodiment of the invention, PRO201, PRO308 or PRO309expression may be reduced by providing PRO201-, PRO308- orPRO309-expressing cells with an amount of PRO201, PRO308 or PRO309antisense RNA or DNA effective to reduce expression of the PRO201,PRO308 or PRO309 protein.

In a further embodiment of the invention, the expression of bindingpartners of PRO201, PRO308 or PRO309 may be reduced by providing PRO201,PRO308 or PRO309 expressing cells with an amount of antisense RNA or DNAeffective for reduced expression of the binding partners of PRO201,PRO308 or PRO309.

I. Diagnostic Uses

Another use of the compounds of the invention (e.g., PRO201, PRO308 orPRO309, PRO201-, PRO308- or PRO309-variants and anti-PRO201, anti-PRO308or anti-PRO309 antibodies) described herein is to help diagnose whethera disorder is driven, to some extent, by PRO201, PRO308 or PRO309modulated signaling.

A diagnostic assay to determine whether a particular disorder is drivenby Nsp1, Nsp2 or Nsp3 signaling, can be carried out using the followingsteps: (1) culturing test cells or tissues; (2) administering a compoundwhich can inhibit Nsp1, Nsp2 or Nsp3 modulated signaling; and (3)measuring the degree of phosphorylation on the PRO201, PRO308 or PRO309substrate in cell lysates or Nsp1, Nsp2 or Nsp3 mediated phenotypiceffects in the test cells. The steps can be carried out using standardtechniques in light of the present disclosure. For example, standardtechniques can be used to isolate cells or tissues and culturing or invivo.

Compounds of varying degree of selectivity are useful for diagnosing therole of PRO201, PRO308 or PRO309. For example, compounds which inhibitNsp 1, Nsp2 or Nsp3 in addition to another form of adaptor molecule canbe used as an initial test compound to determine if one of severaladaptor molecules drive the disorder. The selective compounds can thenbe used to further eliminate the possible role of the other adaptorproteins in driving the disorder. Test compounds should be more potentin inhibiting intracellular signaling activity than in exerting acytotoxic effect (e.g., an IC₅₀ /LD₅₀ of greater than one). The IC₅₀ andLD₅₀ can be measured by stand techniques, such as an MTT assay, or bymeasuring the amount of LDH released. The degree of IC₅₀ /LD₅₀ of acompound should be taken into account in evaluating the diagnosticassay. Generally, the larger the ratio the more relative theinformation. Appropriate controls take into account the possiblecytotoxic effect of a compound of a compound, such as treating cells notassociated with a cell proliferative disorder (e.g., control cells) witha test compound, can also be used as part of the diagnostic assay. Thediagnostic methods of the invention involve the screening for agentsthat modulate the effects of fused upon hedgehog signaling. Exemplarydetection techniques include radioactive labeling andimmunoprecipitating (U.S. Pat. No. 5,385,915).

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manessas, Va.

Example 1

Isolation of cDNA clones Encoding Human PRO201, PRO308 OR PRO309

An expressed sequence tag (EST) DNA database (LIFESEQTM, IncytePharmaceuticals, Palo Alto, Calif.) was searched and an EST wasidentified (1328938, DNA28710, FIG. 4A (SEQ ID NO:13)) which was in afetal pancreas library which shared significant identity which theadaptor protein Shc. A full length cDNA corresponding to the isolatedEST was cloned from a human fetal kidney library using an in vivocloning technique (DNA30676, Nsp1 (SEQ ID NO:2)) in pRK5. There is asingle long open reading frame which encodes a 576 amino acid protein.This native sequence Nsp1 also is related to Sck and Shc (FIG. 8 (SEQ IDNO:17-18, respectively), which is apparent in the SH2 region thatappears at the N-terminus of Nsp1. This native sequence Nsp1 alsocontains a proline-serine rich domain (PS) in the middle of the proteinthat may function as an SH3 interaction domain. The C-terminus of thisnative sequence Nsp1 has no significant identity to any known mammalianproteins. This C-terminal sequence was then used to rescreen the ESTdatabase, wherein was found two additional fragments(104191)(DNA38653)(FIG. 4B) and (1651811)(DNA38654)(FIG. 4C (SEQ IDNO:14-15, respectively)). From these sequences were constructed cloningand enrichment primers, and the corresponding full length sequences wereisolated for native sequence Nsp2 (SEQ ID NO:3) and native sequence Nsp3(SEQ ID NO:5) respectively, using an in vivo cloning technique from ahuman placenta library in pRK5. The probes used for the cloning of thefull length sequences were the following:

    native sequence Nsp1:                                                                                                              Cloning: (SEQ ID                                                             NO:7) ACTGAGGCCTGTTGAA                                                        AGTGCAGAGCTCAG                                                                 Enrichment Primer:                                                           (SEQ ID NO:8)                                                                 GCTGAAGAAGAGCTTCAG                                                              - native sequence                                                           Nsp2:                       Cloning: (SEQ ID NO:9) CAATGCCGATGGCCATTGTGTTGTGTCTTTCAATTATGTCCAGGCGCA       Enrichment Primer: (SEQ ID NO:10) ATCCCAGAATGTCCACTG                           - native sequence Nsp3:                                                      Cloning: (SEQ ID NO:11) GGCCAGCATGATGGACATGGTGTGGAACCTTTCCAGCAGGTCTAGGCG                                                        TA                          Enrichment Primer: (SEQ ID NO:12) GGTGCAGCCCAGGATGTC                    

The three native sequence proteins (Nsp1, Nsp2, Nsp3 (SEQ ID NOS:1, 3,5, respectively) share an overall identity of between 33% and 47% (FIG.6B). Native Sequence Nsp3 (SEQ ID NO:5) has an SH2 domain and apotential SH3 interaction domain (PS region). Native sequence Nsp2 (SEQID NO:3) Nsp2 lacks the SH2 domain but does have a potential SH3interaction domain. The absence of the SH2 domain in native sequenceNsp2 (SEQ ID NO:3) raises the possibility that this protein could act asa dominant negative regulator of the other two Nsps. All three proteinslack apparent kinase or phosphatase domains.

Native sequence cDNA clones Nsp1, Nsp2 and Nsp3 were sequenced in theirentirety. The entire nucleotide sequence of DNA30676, DNA40575 andDNA61601 is shown in FIG. 1 (SEQ ID NO: 2), FIG. 2 (SEQ ID NO: 4) andFIG. 3 (SEQ ID NO: 6), respectively. Clones DNA30676-1223, DNA40575-1223and DNA61601-1223 h been deposited with ATCC and are assigned ATCCdeposit numbers 209567, 209565 and 209713. Moreover, clonesDNA40556-1223 and DNA40554-1223 have also been deposited with the ATCCand are assigned ATCC deposit numbers 209566 and 2096564.

Example 2

Northern Blot Analysis

Expression of PRO201, PRO308 OR PRO309 mRNA in human tissues wasexamined by northern blot analysis. Human RNA blots were hybridized toan ³² P-endlabelled DNA probe complementary to the nucleotide encodingamino acids: (a) 90-102 in DNA30676; (b) 270-284 in DNA40575 or (c)475-491 in DNA6 Endocrine and fetal II (Clontech) were hybridized inExpressHyb® hybridization solution (Clontech) in accordance with themanufacturer's instructions. Blots were incubated with the probes inhybridization buffer (5X SSPE; 2X Denhardt's solution; 100 mglmLdenatured sheared salmon sperm DNA; 50% formamide; 2% SDS) for 16 hoursat 42° C. The blots were washed several times in 2X SSC; 0.05% SDS for 1hour at room temperature, followed by a 30 minute wash in 0.1X SSC; 0.1%SDS at 50° C. The blots were developed after overnight exposure byphosphorimager analysis (Fuji).

As shown in FIG. 9A, significant expression of native sequence Nsp2 (SEQID NO:3) was only detected in human fetal liver and may be expressed inother fetal tissues (e.g., fetal kidney). This pattern of expressionsuggests a role for Nsp1 in coordinating signaling pathways importantfor fetal development. In contrast, native sequence Nsp2 DNA (SEQ IDNO:4) and native sequence Nsp3 DNA (SEQ ID NO:6) were more widelyexpressed in many tissues. In hematopoietic tissues, two Nsp2transcripts (3.8 Kb and 3.2 Kb) were detected (FIG. 9B).

Example 3

EGF, Insulin and Fibronectin induced Nsp1 Phosohorylation and complexformation with p130^(Cas)

As native sequence Nsp1 (SEQ ID NO:1) has three potentiallyphosphorylatable tyrosines, a study was undertaken to determine whetherNsp1 could be phosphorylated in response to a variety of extracellularstimuli. Treatment with EGF induced a rapid tyrosine phosphorylation ofnative sequence Nsp1 (SEQ ID NO:1) which occurred in 2 minutes or less(FIG. 10A). Native sequence Nsp1 (SEQ ID NO:1) is also phosphorylated inresponse to insulin, IGF-1 and heregulin (not shown). In contrast,fibronectin (FN) stimulated only weak Nsp1 phosphorylation (FIG. 10C).

In order to trace the pathway(s) impacted by Nsp1, Applicants haveidentified proteins associated with Nsp1 in vivo by way ofco-immunoprecipitation experiments. Treatment with EGF lead to anassociation between Nsp1 and a tyrosine phosphorylated protein with amolecular mass of approximately 170 kD. This protein is rapidly tyrosinephosphorylated in response to EGF and can be detected with a mABdirected against the EGF receptor. Further, Nsp1 can be detected bywestern blotting following immunoprecipitation of the EGF receptors(FIG. 10B). There is residual Nsp1/EGF receptor interaction prior to EGFtreatment, but the extent of the interaction significantly increasesfollowing exposure to EGF.

The coimmunoprecipitation experiments also revealed that Nsp1 interactswith a 130 kD protein (p130). In serum starved cells p130 wasphosphorylated to a moderate level (FIG. 10A), whereas loss of cellattachment lead to a complete p130 dephosphorylation (FIG. 10C). Bywestern blotting analysis this p130 was found to the adaptor proteinp130^(Cas). In FIG. 10A, anti-(P)Tyr antibody detected two bands atapproximately 130 kD, while anti-Cas antibody recognizes only the bottomband. We have not yet identified the upper band. Cas was originallyfound as a hyper-phosphorylated protein following induced expression ofviral Crk (v-Crk) [Sakai et al., EMBO J. 13: 3748-56 (1994)] and isphosphorylated in response to integrin interaction with extracellularmatrix as well as a number of other stimuli. Chen et al., J. Biol. Chem.272: 27401-10 (1997); Casamassima & Rozengurt, J Biol. Chem. 272:9363-70 (1997); Nojima et al., J. Biol. Chem. 270: 15398-402 (1995). Casdirectly interacts with focal adhesion kinase (FAK)[Polte & Hanks, Proc.Nat. Acad. Sci. USA.92: 10678-82 (1995)] and appears to be a criticalcomponent by which extracellular events influence cell motilitymorphology and survival. Daniel & Reynolds, Mol. & Cell. Biol. 15:4819-24 (1995); Mo & Reynolds, Cancer Res. 56: 2633-40 (1996); Nakamotoet al., Mol. Cell. Biol. 17:3884-97(1997).

The phosphorylation status of Nsp1 and Cas and the relative amount ofCas associated with Nsp1 is dependent on the signaling through eitherthe EGF or integrin receptors. EGF increases Nsp1 phosphorylation butdephosphorylation of both total (data not shown) and Nsp1 associated Cas(FIG. 10A). There is also an increase in the amount of Cas associatedwith Nsp1 after EGF treatment (FIG. 10A). In contrast, fibronectin hadonly a small effect on Nsp1 phosphorylation but increased thephosphorylation of Cas that is associated with Nsp1 and at the same timelead to a transient decrease in the amount of Cas that is associatedwith Nsp1 (FIG. 10C). An increase in Cas phosphorylation in response tointegrins has been previously reported. Nojima et al., J. Biol. Chem.270: 15398-402 (1995). This decrease in the Nsp1/Cas complex reached anadir at approximately 30 minutes and then returned toward baselineconditions at around 4 hours.

In FIG. 11 it is demonstrated that insulin stimulated Nsp1phosphorylation peaked at 2 hours, and then decreased after 14.5 hours.The same blot was reprobed with anti-FLAG antibody to show the equalloading . In FIG. 11 it is demonstrated that IGF-1 also stimulates thephosphorylation of Nsp1 although the level of phosphorylation inresponse to IGF-1 is less than that seen in response to insulin.

IGF-1 results:

In the absence of insulin (FIG. 13) or EGF (not shown) Nsp1 isassociated with Cas. The phosphorylaion of Cas was observed to decreaseafter insulin treatment. This is indicated both in FIG. 12 and FIG. 13A.The membrane of the tested samples in FIG. 13A was stripped and reprobedwith anti-p130^(Cas) antibody to also demonstrate that the amount of Casassociated with Nsp1 decreases following insulin treatment.

Materials and Methods:

EGF/fibronectin:

Transfected and serum starved COS (A,B) were either treated with 25ng/ml EGF for the times indicated or left untreated. Transfected andserum starved 293 cells were either attached to plastic (on dish), heldin suspension (off dish) or replated onto 10 mg/ml FN-coated dishes forthe times indicated FIG. 10C(C). In FIGS. 10A and 10C anti-flagimmunoprecipitates were blotted with anti-flag, anti-(P)Tyr or anti-Casantibodies as indicated. In FIG. 10B, anti-EGF receptor (CalBiochem)immunoprecipitates were blotted with anti-flag or anti-(P)Tyr antibodiesas indicated. Transfected cells were lysed in coimmunoprecipitationassay (ColPA) buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X-100,2 mM EDTA, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mMorthovanadate) containing freshly added protease inhibitors (ImM AEBSF,10 mM leupeptin, 2 mg/ml aprotinin, 1 mM pepstatin). Anti-flag (Kodak,IBI immunoprecipitates and the associated proteins were visualized byanti-(P)Tyr antibody PY20 (Transduction Lab). The same blots werestriped and reblotted with anti-p130^(Cas) (Transduction Lab) oranti-flag antibody and detected with the ECL system (Pierce). The Flapepitope (DYKDDDDK) (SEQ ID NO:19) was added in frame to the N-terminusof the Nsp1 cDNA construct using in vitro mutagenesis to reatepRK.Nsp1.FLAG.

Insulin:

The FLAG epitope (DYKDDDDK) (SEQ ID NO:19) was inserted into theN-terminus of the Nsp1 cDNA construct using a standard in vitromutagenesis to create pRK.Nsp.FLAG. CHO cells overexpressing insulinreceptor (CHO-IR) were cultured in F12-DMEM containing 10% serum, 2 mML-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin.Liposome-mediated transfection methods using DOSPER (BoehringerMannheim) or superfect (Qiagen) were carried out on CHO cells inaccordance with the manufacturers instructions. CHO-JR cells weretransiently transfected with either the empty vector pRK or withpRK.Nsp1.FLAG and serum starved for 16 hours. Cells were treated with orwithout 100 nM insulin for different times and then lysed on ice for onehour in 1 ml of immunoprecipitation assay (IPA) buffer (10 mM Tris,pH7.5, 150 mM CaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mMEGTA, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mMorthovanadate) containing fresh added protease inhibitors (1 mM AEBSF,10 μM leupeptin, 2 μg/ml aprotinin, 1 μM pepstatin). Samples wereimmunoprecipitated with an anti-FLAG affinity gel (IBI, Kodak).Following SDS-polyacrylamide gel electrophoresis, proteins weretransferred onto nitrocellulose membrane (Novex), western blotted withthe anti-phosphotyrosine antibody PY-20 (Transduction Lab) or anti-FLAGantibodies and detected with the ECL system (Pierce).

IGF-1

pRK or pRK.Nsp1.FLAG transfected with CHO-IR or CHO-IGF1R (IGF-1receptor) cells were serum starved, treated with 100 nM insulin or 100ng/ml IGF-1 and lysed in coimmunoprecipitation assay (ColPA) buffer (20mM Tris, pH 7.5, 100 mM NaCI, 1% Triton X-100, 2 mM EDTA, 10 mM sodiumpyrophosphate, 10 mM sodium fluoride, 2 mM orthovanadate) containingprotease inhibitors. Samples were immunoprecipitated with anti-FLAG oranti-p130^(Cas) and Western blotted with the anti-phosphotyrosineantibody PY-20 or anti-p130^(Cas) (Santa Cruz Biotechnology).

Example 4

Mapping of Phosphorylated Tyrosine Residues in Nsp1

In order to map the phosphorylated residues in native sequence Nsp1 (SEQID NO:1), Applicants have independently changed each of the threetyrosine in Nsp1 (SEQ ID NO:1) to phenylalanine. Transfected cells werethen stimulated with EGF and the Nsp1 (SEQ ID NO:1) immunoprecipitated.In all three cases the non-phosphorylated Nsp1 (SEQ ID NO:1)immunoprecipitated from non-stimulated cells was associated with bothCas and the EGF receptor. These results demonstrate that the amino acidchanges were not grossly deleterious to the overall protein structure.While mutant Nsp1_(Y61F) was phosphorylated normally in response to EGF,phosphorylation of Nsp1_(Y95F) was not detected and Nsp1_(Y231F) wasweakly phosphorylated. This data suggests that there is first aphosphorylation of Y95 followed by the phosphorylation of Y231. Y16 mayor may not be phosphorylated, but is not required for phosphorylation ofeither Y95 or Y231. Further, as the amount of EGF receptorcoimmunoprecipitated with native sequence Nsp1 (SEQ ID NO:1) isincreased by receptor phosphorylation but largely independent of Nsp1phosphorylation, it would appear that Nsp1 association with Cas isindependent of both Nsp1 and Cas phosphorylation status (FIG. 10), thisinteraction may be mediated through the SH3 domain of Cas and SH3interaction domain of Nsp1.

Materials and Methods:

All three tyrosine residues in Nsp1 were changed to phenylalanine usinga standard in vitro mutagenesis technique. Mutants (Y16F, Y95F andY231F) and wild type native sequence Nsp1 (SEQ ID NO:1) were transfectedinto COS cells and treated with 25 ng/ml of EGF for 10 min. or leftuntreated. Cell lysates were immunoprecipitated with the anti-flagantibody and Western blotted with either the anti-P(Tyr) antibody, theanti-p 1 ₃₀ cr antibody or the anti-flag antibody.

Example 5

Transformation and Tumorigenicity in Nude Mice

Intro:

Since Cas has been implicated in c-src mediated events [Sakai et al.,EMBO J. 13: 3748-56 (1994); Sakai et al., Oncogene 14: 1419-26 (1997)],Applicants examined the effect of native sequence Nsp1 (SEQ ID NO:1) inan NIH3T3 transformation assay. The NIH3T3 is a cell line which normallygrow in monolayer even when the cells are overconfluent. They may beused to determine whether or not a candidate gene has the potential foroncogenicity when the candidate is transfected via retroviral mediatedinfection in vector MSCV. The transfected cells are allowed to generateinto foci, picked and cultured to 10 million cells and injected intonude mice. It the transfected gene is oncogenic, it will grow onuninhibited by the deficient immune system of the nude mouse and form atumor. See Winograd et al., In Vivo 1 (1): 1-13 (1987).

Discussion and Results:

More than one hundred foci of morphologically transformed cells wereobserved on one 100 mm plate of NIH3T3 cells following transfection witha retrovirus expressing native sequence Nsp1 (SEQ ID NO:1) and G418selection, but none appeared in control (neo) vector. The transformedcells (FIG. 14B) were more rounded and compacted in comparison to thenormal elongated fibroblast shape of the control transfected NIH3T3cells (FIG. 14A). To investigate whether the transformned nativesequence Nsp1 (SEQ ID NO:1) expressing cells were also tumorigenic,three independent foci were picked and expanded to generateNlH3T3-MSCV.Nsp1-.sub1, -.sub2 and -.sub3. Controls consisted of celllines expressing neo only (NIH3T3-neo) and a pool of transfected cellsthat expressed lower levels of native sequence Nsp1 (SEQ ID NO:1) butwas not transformed (NIH3T3-Nsp1.non-trans). The native sequence Nsp1(SEQ ID NO:1) expressing, non-transformed cells were derived byinfecting NIH3T3 cells with the Nsp1 expressing retroviral vector. Thesebulk cultures were selected for neomycin resistance, but were notallowed to proceed through the postconfluent growth that selects forfoci formation.

Each cell line was injected into five mice. No tumor growth was observedin any mice injected with neo control cells or NIH3T3-Nsp1.non-trans.cells. All five mice in each group injected with NIH3T3-MSCV.Nsp1-.sub1,-.sub2, -.sub3 grew obvious tumors within three weeks. Histologicalanalysis indicated that the tumors consisted of large, irregular,moderately anaplastic epithelioid cells with a high mitotic index (FIG.14C). There was no evidence of metastasis.

The tumors which formed were well circumscribed, locally expansilemasses composed primarily of larger, irregular, moderately anaplasticepitheloid cells with a high mitotic index, interspersed, peripherallyby small areas of spindle-cell proliferation. (FIG. 14C).

Materials and Methods:

pRK.Nsp1.FLAG plasmid was digested with EcoRI and Sal I. The nativesequence Nsp1 (SEQ ID NO:1) cDNA fragment including the FLAG epitope waspurified and subcloned into the EcoRI and Xho I sites of the retroviralvector MSCVneo resulting in MSCVneo.Nsp1.FLAG. Mouse embryonicfibroblast cells (ATCC) and retroviral producer BOSC 23 cells weremaintained in DMEM with 10% fetal bovine serum, 2 mM L-glutamine, 100units/ml penicillin, and 100 μg/ml streptomycin.

The retroviral vectors MSCVneo and MSCVneo.Nsp1 FLAG were transfectedinto BOSC 23 cells using calcium phosphate-mediated transfection. The72-hour supernatant was used to infect NIH3T3 cells plated on a 6-wellplate. Infected cells were selected in 400 μg/ml G418 (Gibco) and pooledto generate NIH3T3-MSCV and -MSCV.Nsp1 cell lines. NIH3T3-MSCV and-MSCV.Nsp1 cells were grown until confluent for 4 days with a mediumchange once, split at a one to five ratio and grown until confluent foranother 4 days. More than one hundred foci of morphologicallytransformed cells were observed on one 100 mm plate of NIH3T3 cellsfollowing infection, but none in the control (neo) vector. Foci weresubjected to ultrathin section followed by Tuluidine blue staining. FIG.14A shows ultrathin sections of either control cells (FIG. 14A), whileFIG. 14B shows native sequence Nsp1 (SEQ ID NO:1) transformed foci.

Three transformed foci of NIH3T3-MSCV.Nsp1 were picked and expanded togenerate sublines NIH3T3-MSCV.Nsp1-sc1, -sc2 and -sc3. 10⁷ vectortransfected control cells, untransformed NIH3T3.MSCV.Nsp1 and threesublines were injected subcutaneously into the back of each nude mouse.Five mice were injected for each cell line.

Tumor mass was measured at two weeks and four weeks. The resultanttumors (four weeks post injection) were fixed, blocked and sectionsstained with hematoxylin and eosin. (FIG. 14C). No tumor growth wasobserved in any mouse injected with vector transfected control cells oruntransformed NIH3T3-MSCV.Nsp1 cells. Every mouse injected withNIH3T3-MSCV.Nsp1-.sc1, -.sc2 or -.sc3 grew tumors. (FIG. 15).

Example 6

Apoptosis Resistance

Since the previous examples indicated that Nsp1 expression leads toNIH3T3 transformation and tumor formation, Applicants have investigatedwhether native sequence Nsp1 (SEQ ID NO:1) expression protects cellsfrom apoptosis induced by removal of growth factors. Subcloned celllines were derived from the morphologically transformed cellsNIH3T3-MSCV.Nsp1-.sub1 and -.sub2 (designated Nsp1.-sub1.1 andNsp1.sub2.1). These transformed clonal lines, the non-transformed cellculture (NIH3T3-Nsp1.non-trans.) and the control cells NIH3T3-neo wereserum starved for 48 hours in the presence or absence of the PI 3-kinaseinhibitor LY294002 and subjected to ANNEXIN V (Clontech) apoptosis assay(FIG. 16). Although the NIH3T3-Nsp1.non-trans. cells weremorphologically normal and did not form tumors in nude mice they weremore resistant to apoptosis induced by growth factor withdrawal thanwere the control NIH3T3-neo cells. This small but significant increasein resistance to apoptosis was abolished by the P13 kinase inhibitorLY294002. In the vector control cells which do not express nativesequence Nsp1 (SEQ ID NO:1), LY294002 did not by itself induce furtherapoptosis. In the Nsp1 transformed sublines, there was an almostcomplete protection from serum starvation induced apoptosis, but thiseffect was not sensitive to the treatment with the PI 3-kinaseinhibitor. This dependent on PI 3-kinase at lower levels of Nsp1 wouldplace PI 3-kinase downstream of native sequence Nsp1 (SEQ ID NO:1). Incontrast the observation that the growth factor independence at highNsp1 levels is not inhibitable by LY294002 suggests that Nsp1 impacts anadditional pathway that functions independently of PI 3-kinase. That PI3-kinase is both necessary and sufficient for growth factor mediatedresistance to apoptosis had been previously reported. Kulik et al., Mol.Cell Biol. 17: 1595-606 (1997); Parrizas et al., J. Biol. Chem. 272:154-61 (1997); Vemuri et al., Development 122: 2529-37 (1996).

Material and Methods:

Control cells (NIH3T3-neo), non-transformed Nsp1 expressing cells(NIH3T3-Nsp1.non-trans.) and the transformed sublines (Nsp1₁.1 andNsp1.sub2.1) were serum starved in the presence of absence of 10 μg/mlLY294002 for 48 hours. The percent of apoptotic cells were assayed usingANNEXIN V-FITC (Clontech) on FACS according to the manufacturersdirections. Each cell line was assayed in triplicate and the means andstandard deviations are shown. In transformed sublines, native sequenceNsp1 (SEQ ID NO:1) protected cells from serum starvation inducedapoptosis.

Example 7

PI 3-Kinase Interaction

In order to determine whether Nsp1 does interact with PI 3-kinase, a GSTfusion protein containing the PI 3-kinase N-terminal or C-terminal SH2domains were incubated with EGF treated or untreated COS cell lysatetransiently expressing native sequence Nsp1 (SEQ ID NO:1) or controls(FIG. 20). The C-terminal SH2 domain GST fusion protein does interactwith native sequence Nsp1 (SEQ ID NO:1). This interaction appears to beat least partially dependent on the phosphorylation status of nativesequence Nsp1 (SEQ ID NO:1) as there is an increase in the amount ofNsp1 that interacts with P13 kinase following EGF stimulation. TheN-terminal SH2 domain of P13 kinase does not measureably interact withnative sequence Nsp1 (SEQ ID NO:1).

Material and Methods:

COS cells transfected with pRK or with native sequence Nsp1 (SEQ IDNO:1) were treated with 25 ng/ml EGF or left untreated. Cells were lysedin CoIP buffer (supra) and incubated with PI 3-kinase N-terminal orC-terminal SH2 domain-GST beads (UBI). The precipitated Nsp1 wasdetected with anti-flag antibody.

Example 8

Expression of PRO201, PRO308 or PRO309 in E. coli

This example illustrates preparation of an unglycosylated form ofPRO201, PRO308 or PRO309 by recombinant expression in E. coli.

The DNA sequence encoding PRO201, PRO308 or PRO309 (SEQ ID NO:2, SEQ IDNO: 4, SEQ ID NO: 6) is initially amplified using selected PCR primers.The primers should contain restriction enzyme sites which correspond tothe restriction enzyme sites on the selected expression vector. Avariety of expression vectors may be employed. An example of a suitablevector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95(1977)) which contains genes for ampicillin and tetracycline resistance.The vector is digested with restriction enzyme and dephosphorylated. ThePCR amplified sequences are then ligated into the vector. The vectorwill preferably include sequences which encode for an antibioticresistance gene, a trp promoter, a polyhis leader (including the firstsix STII codons, polyhis sequence, and enterokinase cleavage site), thePRO201, PRO308 or PRO309 coding region, lambda transcriptionalterminator, and an argu gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO201, PRO308 or PRO309 protein can then bepurified using a metal chelating column under conditions that allowtight binding of the protein.

Example 9

Expression of PRO201, PRO308 orPRO309 in mammalian cells

This example illustrates preparation of a glycosylated form of PRO201,PRO308 or PRO309 by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. optionally, the PRO201, PRO308 or PRO309 DNAis ligated into pRK5 with selected restriction enzymes to allowinsertion of the PRO201, PRO308 or PRO309 DNA using ligation methodssuch as described in Sambrook et al., supra. The resulting vector iscalled pRK5-PRO201, pRK5-PRO308 or pRK5-PRO309.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO201, pRK5-PRO308 or pRK5-PRO309 DNA is mixed with about 1 μg DNAencoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] anddissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. Tothis mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mMNaCl, 1.5 mM NaPO₄, and a precipitate is allowed to form for 10 minutesa 25° C. The precipitate is suspended and added to the 293 cells andallowed to settle for about four hours at 37° C. The culture medium isaspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds.The 293 cells are then washed with serum free medium, fresh medium isadded and the cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵ S-cysteine and 200 μCi/ml ³⁵ S-methionine.After a 12 hour incubation, the conditioned medium is collected,concentrated on a spin filter, and loaded onto a 15% SDS gel. Theprocessed gel may be dried and exposed to film for a selected period oftime to reveal the presence of PRO201, PRO308 or PRO309 polypeptide. Thecultures containing transfected cells may undergo further incubation (inserum free medium) and the medium is tested in selected bioassays.

In an alternative technique, PRO201, PRO308 or PRO309 may be introducedinto 293 cells transiently using the dextran sulfate method described bySomparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells aregrown to maximal density in a spinner flask and 700 ug pRK5-PRO201,pRK5-PRO308 or pRK5-PRO309 DNA is added. The cells are firstconcentrated from the spinner flask by centrifugation and washed withPBS. The DNA-dextran precipitate is incubated on the cell pellet forfour hours. The cells are treated with 20% glycerol for 90 seconds,washed with tissue culture medium, and re-introduced into the spinnerflask containing tissue culture medium, 5 :g/ml bovine insulin and 0.1:g/ml bovine transferrin. After about four days, the conditioned mediais centrifuged and filtered to remove cells and debris. The samplecontaining expressed PRO201, PRO308 or PRO309 can then be concentratedand purified by any selected method, such as dialysis and/or columnchromatography.

In another embodiment, PRO201, PRO308 or PRO309 can be expressed in CHOcells. The pRK5-PRO201, PRO308 or PRO309 can be transfected into CHOcells using known reagents such as CaPO₄ or DEAE-dextran. As describedabove, the cell cultures can be incubated, and the medium replaced withculture medium (alone) or medium containing a radiolabel such as ³⁵S-methionine. After determining the presence of PRO201, PRO308 or PRO309polypeptide, the culture medium may be replaced with serum free medium.Preferably, the cultures are incubated for about 6 days, and then theconditioned medium is harvested. The medium containing the expressedPRO201, PRO308 or PRO309 can then be concentrated and purified by anyselected method.

Epitope-tagged PRO201, PRO308 or PRO309 may also be expressed in hostCHO cells. The PRO201, PRO308 or PRO309 may be subcloned out of the pRK5vector. The subclone insert can undergo PCR to fuse in frame with aselected epitope tag such as a poly-his tag into a Baculovirusexpression vector. The poly-his tagged PRO201, PRO308 or PRO309 insertcan then be subcloned into a SV40 driven vector containing a selectionmarker such as DHFR for selection of stable clones. Finally, the CHOcells can be transfected (as described above) with the SV40 drivenvector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedPRO201, PRO308 or PRO309 can then be concentrated and purified by anyselected method, such as by Ni²⁺ -chelate affinity chromatography.

Example 10

Expression of PRO201, PRO308 or PRO309 in yeast.

The following method describes recombinant expression of PRO201, PRO308or PRO309 in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO201, PRO308 or PRO309 from the ADH2/GAPDHpromoter. DNA encoding PRO201, PRO308 or PRO309, a selected signalpeptide and the promoter is inserted into suitable restriction enzymesites in the selected plasmid to direct intracellular expression ofPRO201, PRO308 or PRO309. For secretion, DNA encoding PRO201, PRO308 orPRO309 can be cloned into the selected plasmid, together with DNAencoding the ADH2/GAPDH promoter, the yeast alpha-factor secretorysignal/leader sequence, and linker sequences (if needed) for expressionof PRO201, PRO308 or PR0309.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO201, PRO308 or PRO309 can subsequently be isolated andpurified by removing the yeast cells from the fermentation medium bycentriftigation and then concentrating the medium using selectedcartridge filters. The concentrate containing PRO201, PRO308 or PRO309may further be purified using selected column chromatography resins.

Example 11

Expression of PRO201, PRO308 or PRO309 in Baculovirus-Infected InsectCells

The following method describes recombinant expression of PRO201, PRO308or PRO309 in Baculovirus-infected insect cells.

The PRO201, PRO308 or PRO309 is fused upstream of an epitope tagcontained with a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thePRO201, PRO308 or PRO309 or the desired portion of the PRO201, PRO308 orPRO309 (such as the sequence encoding the extracellular domain of atransmembrane protein) is amplified by PCR with primers complementary tothe 5' and 3' regions. The 5' primer may incorporate flanking (selected)restriction enzyme sites. The product is then digested with thoseselected restriction enzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodoplerafrugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression is performed as described byO'Reilley et al., Baculovirus expression vectors: A laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO201, PRO308 or PRO309 can then be purified,for example, by Ni²⁺ -chelate affinity chromatography as follows.Extracts are prepared from recombinant virus-infected Sf9 cells asdescribed by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9;12.5 mM MgCl₂ ; 0.1 mM EDTA; 10% Glycerol; 0.1% NP40; 0.4 M KCl), andsonicated twi 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filteredthrough a 0.45 Fm filter. A Ni²⁺ -agarose column (commercially availablefrom Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL ofwater and equilibrated with 25 mL of loading buffer. The filtered cellextract is loaded onto the column at 0.5 mL per minute. The column iswashed to baseline A₂₈₀ with loading buffer, at which point fractioncollection is started. Next, the column is washed with a secondary washbuffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH 6.0), whichelutes nonspecifically bound protein. After reaching A₂₈₀ baselineagain, the column is developed with a 0 to 500 mM Imidazole gradient inthe secondary wash buffer. One mL fractions are collected and analyzedby SDS-PAGE and silver staining or western blot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containingthe eluted His₁₀ -tagged PRO201, PRO308 or PRO309 are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO201,PRO308 or PRO309 can be performed using known chromatography techniques,including for instance, Protein A or protein G column chromatography.

Example 12

Preparation of Antibodies that Bind PRO201, PRO308 or PRO309

This example illustrates preparation of monoclonal antibodies which canspecifically bind PRO201, PRO308 or PRO309.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO201, PRO308 or PRO309, fusion proteinscontaining PRO201, PRO308 or PRO309, and cells expressing recombinantPRO201, PRO308 or PRO309 on the cell surface. Selection of the immunogencan be made by the skilled artisan without undue experimentation.

Mice, such as Balb/c, are immunized with the PRO201, PRO308 or PRO309immunogen emulsified in complete Freund's adjuvant and injectedsubcutaneously or intraperitoneally in an amount from 1-100 micrograms.Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (RibiImmunochemical Research, Hamilton, Mt.) and injected into the animal'shind foot pads. The immunized mice are then boosted 10 to 12 days laterwith additional immunogen emulsified in the selected adjuvant.Thereafter, for several weeks, the mice may also be boosted withadditional immunization injections. Serum samples may be periodicallyobtained from the mice by retro-orbital bleeding for testing in ELISAassays to detect PRO201, PRO308 or PRO309 antibodies.

After a suitable antibody titer has been detected, the animals"positive" for antibodies can be injected with a final intravenousinjection of PRO201, PRO308 or PRO309. Three to four days later, themice are sacrificed and the spleen cells are harvested. The spleen cellsare then fused (using 35% polyethylene glycol) to a selected murinemyeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL 1597.The fusions generate hybridoma cells which can then be plated in 96 welltissue culture plates containing HAT (hypoxanthine, aminopterin, andthymidine) medium to inhibit proliferation of non-fused cells, myelomahybrids, and spleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstPRO201, PRO308 or PRO309. Determination of "positive" hybridoma cellssecreting the desired monoclonal antibodies against PRO201, PRO308 orPRO309 is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PRO201,anti-PRO308 or anti-PRO309 monoclonal antibodies. Alternatively, thehybridoma cells can be grown in tissue culture flasks or roller bottles.Purification of the monoclonal antibodies produced in the ascites can beaccomplished using ammonium sulfate precipitation, followed by gelexclusion chromatography. Alternatively, affinity chromatography basedupon binding of antibody to protein A or protein G can be employed.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):

    ______________________________________                                        Material       ATCC Dep. No. Deposit Date                                     ______________________________________                                        DNA30676-1223  209567        12/23/97                                           DNA40575-1223 209565 12/23/97                                                 DNA40556-1223 209566 12/23/97                                                 DNA40554-1223 209564 12/23/97                                                 DNA61601-1223 209713 3/31/98                                                ______________________________________                                    

DNA40556 & DNA40554 may be combined to cover a full length codingsequence of an Nsp3 variant.

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC § 122 and the Commissioner's rules pursuantthereto (including 37 CFR § 1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 19                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 576                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 1                                                         - - Met Gln Val Pro Gln Asp Gly Glu Asp Leu Al - #a Gly Gln Pro Trp           1               5 - #                 10 - #                 15              - - Tyr His Gly Leu Leu Ser Arg Gln Lys Ala Gl - #u Ala Leu Leu Gln                           20 - #                 25 - #                 30              - - Gln Asp Gly Asp Phe Leu Val Arg Ala Ser Gl - #y Ser Arg Gly Gly                           35 - #                 40 - #                 45              - - Asn Pro Val Ile Ser Cys Arg Trp Arg Gly Se - #r Ala Leu His Phe                           50 - #                 55 - #                 60              - - Glu Val Phe Arg Val Ala Leu Arg Pro Arg Pr - #o Gly Arg Pro Thr                           65 - #                 70 - #                 75              - - Ala Leu Phe Gln Leu Glu Asp Glu Gln Phe Pr - #o Ser Ile Pro Ala                           80 - #                 85 - #                 90              - - Leu Val His Ser Tyr Met Thr Gly Arg Arg Pr - #o Leu Ser Gln Ala                           95 - #                100 - #                105              - - Thr Gly Ala Val Val Ser Arg Pro Val Thr Tr - #p Gln Gly Pro Leu                          110  - #               115  - #               120              - - Arg Arg Ser Phe Ser Glu Asp Thr Leu Met As - #p Gly Pro Ala Arg                          125  - #               130  - #               135              - - Ile Glu Pro Leu Arg Ala Arg Lys Trp Ser As - #n Ser Gln Pro Ala                          140  - #               145  - #               150              - - Asp Leu Ala His Met Gly Arg Ser Arg Glu As - #p Pro Ala Gly Met                          155  - #               160  - #               165              - - Glu Ala Ser Thr Met Pro Ile Ser Ala Leu Pr - #o Arg Thr Ser Ser                          170  - #               175  - #               180              - - Asp Pro Val Leu Leu Lys Ala Pro Ala Pro Le - #u Gly Thr Val Ala                          185  - #               190  - #               195              - - Asp Ser Leu Arg Ala Ser Asp Gly Gln Leu Gl - #n Ala Lys Ala Pro                          200  - #               205  - #               210              - - Thr Lys Pro Pro Arg Thr Pro Ser Phe Glu Le - #u Pro Asp Ala Ser                          215  - #               220  - #               225              - - Glu Arg Pro Pro Thr Tyr Cys Glu Leu Val Pr - #o Arg Val Pro Ser                          230  - #               235  - #               240              - - Val Gln Gly Thr Ser Pro Ser Gln Ser Cys Pr - #o Glu Pro Glu Ala                          245  - #               250  - #               255              - - Pro Trp Trp Glu Ala Glu Glu Asp Glu Glu Gl - #u Glu Asn Arg Cys                          260  - #               265  - #               270              - - Phe Thr Arg Pro Gln Ala Glu Ile Ser Phe Cy - #s Pro His Asp Ala                          275  - #               280  - #               285              - - Pro Ser Cys Leu Leu Gly Pro Gln Asn Arg Pr - #o Leu Glu Pro Gln                          290  - #               295  - #               300              - - Val Leu His Thr Leu Arg Gly Leu Phe Leu Gl - #u His His Pro Gly                          305  - #               310  - #               315              - - Ser Thr Ala Leu His Leu Leu Leu Val Asp Cy - #s Gln Ala Thr Gly                          320  - #               325  - #               330              - - Leu Leu Gly Val Thr Arg Asp Gln Arg Gly As - #n Met Gly Val Ser                          335  - #               340  - #               345              - - Ser Gly Leu Glu Leu Leu Thr Leu Pro His Gl - #y His His Leu Arg                          350  - #               355  - #               360              - - Leu Glu Leu Leu Glu Arg His Gln Thr Leu Al - #a Leu Ala Gly Ala                          365  - #               370  - #               375              - - Leu Ala Val Leu Gly Cys Ser Gly Pro Leu Gl - #u Glu Arg Ala Ala                          380  - #               385  - #               390              - - Ala Leu Arg Gly Leu Val Glu Leu Ala Leu Al - #a Leu Arg Pro Gly                          395  - #               400  - #               405              - - Ala Ala Gly Asp Leu Pro Gly Leu Ala Ala Va - #l Met Gly Ala Leu                          410  - #               415  - #               420              - - Leu Met Pro Gln Val Ser Arg Leu Glu His Th - #r Trp Arg Gln Leu                          425  - #               430  - #               435              - - Arg Arg Ser His Thr Glu Ala Ala Leu Ala Ph - #e Glu Gln Glu Leu                          440  - #               445  - #               450              - - Lys Pro Leu Met Arg Ala Leu Asp Glu Gly Al - #a Gly Pro Cys Asp                          455  - #               460  - #               465              - - Pro Gly Glu Val Ala Leu Pro His Val Ala Pr - #o Met Val Arg Leu                          470  - #               475  - #               480              - - Leu Glu Gly Glu Glu Val Ala Gly Pro Leu As - #p Glu Ser Cys Glu                          485  - #               490  - #               495              - - Arg Leu Leu Arg Thr Leu His Gly Ala Arg Hi - #s Met Val Arg Asp                          500  - #               505  - #               510              - - Ala Pro Lys Phe Arg Lys Val Ala Ala Gln Ar - #g Leu Arg Gly Phe                          515  - #               520  - #               525              - - Arg Pro Asn Pro Glu Leu Arg Glu Ala Leu Th - #r Thr Gly Phe Val                          530  - #               535  - #               540              - - Arg Arg Leu Leu Trp Gly Ser Arg Gly Ala Gl - #y Ala Pro Arg Ala                          545  - #               550  - #               555              - - Glu Arg Phe Glu Lys Phe Gln Arg Val Leu Gl - #y Val Leu Ser Gln                          560  - #               565  - #               570              - - Arg Leu Glu Pro Asp Arg                                                                  575 5 - #76                                                    - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 2413                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 2                                                         - - cgggggtgac agcagcccgg agccgcggag cctcagcttc cgcctggacc  - #                  50                                                                         - - cagcctcgtg ggagccccgc gggtcctgcc cagatgtgga agactgaggc  - #                 100                                                                         - - ctgttgaaag tgcagagctc agccctggca ccctctgttc ccaagagctc  - #                 150                                                                         - - c   atg cag gtg cca cag gat gga gaa gac - # ctt gct ggc                 - #     187                                                                        Met Gln Val Pro Gln Asp Gly Glu - #Asp Leu Ala Gly                              1            - #   5               - #   10                              - - caa ccc tgg tac cac ggc ctc ctg tcc cgc ca - #g aag gct                  - #    226                                                                    Gln Pro Trp Tyr His Gly Leu Leu Ser Arg Gl - #n Lys Ala                                15         - #         20         - #         25                      - - gaa gct ctt ctt cag caa gat ggc gac ttc ct - #g gtt cgc                  - #    265                                                                    Glu Ala Leu Leu Gln Gln Asp Gly Asp Phe Le - #u Val Arg                                        30 - #                 35                                     - - gcc tct ggg tcc cgt ggg ggc aac ccc gtg at - #c tcc tgc                  - #    304                                                                    Ala Ser Gly Ser Arg Gly Gly Asn Pro Val Il - #e Ser Cys                            40             - #     45             - #     50                          - - cgc tgg cgg ggc tca gcc ctc cat ttt gag gt - #g ttc cgt                  - #    343                                                                    Arg Trp Arg Gly Ser Ala Leu His Phe Glu Va - #l Phe Arg                                    55     - #             60                                         - - gtg gcc ctg cgt ccc cgg cca ggc cga ccc ac - #a gcc ctc                  - #    382                                                                    Val Ala Leu Arg Pro Arg Pro Gly Arg Pro Th - #r Ala Leu                        65                 - # 70                 - # 75                              - - ttt caa ctg gag gat gag caa ttc ccc agc at - #a ccg gct                  - #    421                                                                    Phe Gln Leu Glu Asp Glu Gln Phe Pro Ser Il - #e Pro Ala                                80         - #         85         - #         90                      - - ctg gtt cac agt tat atg aca ggc agg cgc cc - #a ctg tcc                  - #    460                                                                    Leu Val His Ser Tyr Met Thr Gly Arg Arg Pr - #o Leu Ser                                        95 - #                100                                     - - cag gcc aca ggg gct gtg gtc tcc agg cct gt - #g act tgg                  - #    499                                                                    Gln Ala Thr Gly Ala Val Val Ser Arg Pro Va - #l Thr Trp                           105              - #   110              - #   115                          - - cag ggg cct ctg cga cgc agc ttt agc gag ga - #c acc ctg                  - #    538                                                                    Gln Gly Pro Leu Arg Arg Ser Phe Ser Glu As - #p Thr Leu                                   120      - #           125                                         - - atg gat ggc cca gct cgg ata gag cct ctc ag - #g gca agg                  - #    577                                                                    Met Asp Gly Pro Ala Arg Ile Glu Pro Leu Ar - #g Ala Arg                       130                 1 - #35                 1 - #40                            - - aag tgg agc aac agt cag cct gca gat ttg gc - #a cat atg                  - #    616                                                                    Lys Trp Ser Asn Ser Gln Pro Ala Asp Leu Al - #a His Met                               145          - #       150          - #       155                      - - ggg cgg tca aga gaa gac ccc gct ggg atg ga - #a gcc tcc                  - #    655                                                                    Gly Arg Ser Arg Glu Asp Pro Ala Gly Met Gl - #u Ala Ser                                       160  - #               165                                     - - acc atg ccc ata tct gcc ttg ccc cga acg ag - #c agt gac                  - #    694                                                                    Thr Met Pro Ile Ser Ala Leu Pro Arg Thr Se - #r Ser Asp                           170              - #   175              - #   180                          - - ccg gtg ttg ctg aag gcc cct gct ccc ctg gg - #a act gtt                  - #    733                                                                    Pro Val Leu Leu Lys Ala Pro Ala Pro Leu Gl - #y Thr Val                                   185      - #           190                                         - - gcc gac agt ctc agg gcc tcc gat ggg cag ct - #t caa gcc                  - #    772                                                                    Ala Asp Ser Leu Arg Ala Ser Asp Gly Gln Le - #u Gln Ala                       195                 2 - #00                 2 - #05                            - - aag gca cca acg aag ccc ccc cgg aca ccc tc - #c ttc gaa                  - #    811                                                                    Lys Ala Pro Thr Lys Pro Pro Arg Thr Pro Se - #r Phe Glu                               210          - #       215          - #       220                      - - ctg cct gat gcc tct gaa cgt ccc ccg acg ta - #c tgc gag                  - #    850                                                                    Leu Pro Asp Ala Ser Glu Arg Pro Pro Thr Ty - #r Cys Glu                                       225  - #               230                                     - - ctg gtg ccc cga gtg ccc agt gtc cag gga ac - #a tcc ccg                  - #    889                                                                    Leu Val Pro Arg Val Pro Ser Val Gln Gly Th - #r Ser Pro                           235              - #   240              - #   245                          - - agc caa agc tgc cca gag cca gag gcc cca tg - #g tgg gag                  - #    928                                                                    Ser Gln Ser Cys Pro Glu Pro Glu Ala Pro Tr - #p Trp Glu                                   250      - #           255                                         - - gcc gag gag gat gag gag gaa gag aat aga tg - #t ttt aca                  - #    967                                                                    Ala Glu Glu Asp Glu Glu Glu Glu Asn Arg Cy - #s Phe Thr                       260                 2 - #65                 2 - #70                            - - aga cca cag gct gag atc tct ttc tgc ccc ca - #t gat gcc                  - #   1006                                                                    Arg Pro Gln Ala Glu Ile Ser Phe Cys Pro Hi - #s Asp Ala                               275          - #       280          - #       285                      - - ccc tcc tgc ctg ctg ggc ccc cag aat cgg cc - #c ctg gaa                  - #   1045                                                                    Pro Ser Cys Leu Leu Gly Pro Gln Asn Arg Pr - #o Leu Glu                                       290  - #               295                                     - - ccc caa gtc ctg cat acc ctc cgt ggc ctg tt - #c ctg gag                  - #   1084                                                                    Pro Gln Val Leu His Thr Leu Arg Gly Leu Ph - #e Leu Glu                           300              - #   305              - #   310                          - - cac cat cct ggg agc acc gcc ctt cac ctg ct - #a ttg gta                  - #   1123                                                                    His His Pro Gly Ser Thr Ala Leu His Leu Le - #u Leu Val                                   315      - #           320                                         - - gac tgc cag gcc aca ggc ctc ctg gga gtg ac - #c aga gat                  - #   1162                                                                    Asp Cys Gln Ala Thr Gly Leu Leu Gly Val Th - #r Arg Asp                       325                 3 - #30                 3 - #35                            - - cag cgg ggc aac atg gga gtc tca tct ggc ct - #g gag ctg                  - #   1201                                                                    Gln Arg Gly Asn Met Gly Val Ser Ser Gly Le - #u Glu Leu                               340          - #       345          - #       350                      - - ctc act ctt ccc cat gga cac cac ttg agg tt - #g gaa ctg                  - #   1240                                                                    Leu Thr Leu Pro His Gly His His Leu Arg Le - #u Glu Leu                                       355  - #               360                                     - - ctg gag agg cat cag aca ctg gcg ctg gcc gg - #g gcg ctg                  - #   1279                                                                    Leu Glu Arg His Gln Thr Leu Ala Leu Ala Gl - #y Ala Leu                           365              - #   370              - #   375                          - - gcg gtg ctg ggc tgc tcg ggg ccg ctg gag ga - #g cgc gca                  - #   1318                                                                    Ala Val Leu Gly Cys Ser Gly Pro Leu Glu Gl - #u Arg Ala                                   380      - #           385                                         - - gcc gca ctg agg gga ctg gta gag ctg gcg ct - #g gcg ctg                  - #   1357                                                                    Ala Ala Leu Arg Gly Leu Val Glu Leu Ala Le - #u Ala Leu                       390                 3 - #95                 4 - #00                            - - cgg cca ggg gcg gcg ggg gac ctg ccc ggg ct - #g gct gca                  - #   1396                                                                    Arg Pro Gly Ala Ala Gly Asp Leu Pro Gly Le - #u Ala Ala                               405          - #       410          - #       415                      - - gtc atg ggc gcc ctg ctc atg ccc cag gtg tc - #c cgg ttg                  - #   1435                                                                    Val Met Gly Ala Leu Leu Met Pro Gln Val Se - #r Arg Leu                                       420  - #               425                                     - - gag cac acg tgg cgc cag ctc cga agg agc ca - #c acg gag                  - #   1474                                                                    Glu His Thr Trp Arg Gln Leu Arg Arg Ser Hi - #s Thr Glu                           430              - #   435              - #   440                          - - gct gcg ctg gcc ttt gag cag gag ctg aag cc - #g ctg atg                  - #   1513                                                                    Ala Ala Leu Ala Phe Glu Gln Glu Leu Lys Pr - #o Leu Met                                   445      - #           450                                         - - cgg gct ctg gat gag ggc gct gga ccc tgc ga - #c ccc ggc                  - #   1552                                                                    Arg Ala Leu Asp Glu Gly Ala Gly Pro Cys As - #p Pro Gly                       455                 4 - #60                 4 - #65                            - - gag gtg gcg ctg ccg cac gtg gca ccc atg gt - #t cgc cta                  - #   1591                                                                    Glu Val Ala Leu Pro His Val Ala Pro Met Va - #l Arg Leu                               470          - #       475          - #       480                      - - ctg gag ggc gag gaa gtc gcg ggg ccg ctg ga - #c gag agc                  - #   1630                                                                    Leu Glu Gly Glu Glu Val Ala Gly Pro Leu As - #p Glu Ser                                       485  - #               490                                     - - tgt gag cgg ctg ttg cgc acc ctg cac ggg gc - #g cgt cac                  - #   1669                                                                    Cys Glu Arg Leu Leu Arg Thr Leu His Gly Al - #a Arg His                           495              - #   500              - #   505                          - - atg gtc cgg gac gca ccc aaa ttc cgc aag gt - #g gca gcc                  - #   1708                                                                    Met Val Arg Asp Ala Pro Lys Phe Arg Lys Va - #l Ala Ala                                   510      - #           515                                         - - cag cgc ctg cga gga ttc cgg cct aac ccg ga - #g ctg agg                  - #   1747                                                                    Gln Arg Leu Arg Gly Phe Arg Pro Asn Pro Gl - #u Leu Arg                       520                 5 - #25                 5 - #30                            - - gag gcc ctg acc acc ggc ttc gtg cgg agg ct - #g ctc tgg                  - #   1786                                                                    Glu Ala Leu Thr Thr Gly Phe Val Arg Arg Le - #u Leu Trp                               535          - #       540          - #       545                      - - ggt agc cgg ggc gcg gga gct ccg cgc gct ga - #a cgc ttt                  - #   1825                                                                    Gly Ser Arg Gly Ala Gly Ala Pro Arg Ala Gl - #u Arg Phe                                       550  - #               555                                     - - gag aag ttc cag cgc gtc ctc ggc gtc ctg tc - #g cag cgc                  - #   1864                                                                    Glu Lys Phe Gln Arg Val Leu Gly Val Leu Se - #r Gln Arg                           560              - #   565              - #   570                          - - ctg gag cct gac cgc t gagagcgcag acacccttct tcac - #acccgg                  1910                                                                       Leu Glu Pro Asp Arg                                                                       575 576                                                            - - gacccccagg tttttgcgaa ccccagaaga gaccaaagga gtcgtcccag  - #                1960                                                                         - - gctcctcgcg cctcaggtgg aatcctgccc tgtgcctcac agaagaggtg  - #                2010                                                                         - - gggaccgcag tcagggtcac ctggaccatg gtgaacatgt gacctgcaga  - #                2060                                                                         - - tctggcatca gaggccagag ttcaaatgtg actccacctc ttaaaagccg  - #                2110                                                                         - - tgatttctag cagttgactt cacctctgtg tcggccttta acaaaatcat  - #                2160                                                                         - - agccatacag cagctcaggc ctgtaatctc agcactttgg gaggccgagg  - #                2210                                                                         - - cggaaggaag gcttgaggcc aggagttcaa gaccagccag ggcaacatgg  - #                2260                                                                         - - tgagacctca tctctacaaa aactgaaaaa taaaaaactt ttaaaaaatg  - #                2310                                                                         - - taaaaaaaaa aaaaaaaggg cggccgcgac tctagagtcg acctgcagaa  - #                2360                                                                         - - gcttggccgc catggcccaa cttgtttatt gcagcttata atggttacaa  - #                2410                                                                         - - ata                  - #                  - #                  - #               2413                                                                   - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 501                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 3                                                         - - Met Gln Asp Arg Arg Ala Leu Ser Leu Lys Al - #a His Gln Ser Glu            1               5 - #                 10 - #                 15              - - Ser Tyr Leu Pro Ile Gly Cys Lys Leu Pro Pr - #o Gln Ser Ser Gly                           20 - #                 25 - #                 30              - - Val Asp Thr Ser Pro Cys Pro Asn Ser Pro Va - #l Phe Arg Thr Gly                           35 - #                 40 - #                 45              - - Ser Glu Pro Ala Leu Ser Pro Ala Val Val Ar - #g Arg Val Ser Ser                           50 - #                 55 - #                 60              - - Asp Ala Arg Ala Gly Glu Ala Leu Arg Gly Se - #r Asp Ser Gln Leu                           65 - #                 70 - #                 75              - - Cys Pro Lys Pro Pro Pro Lys Pro Cys Lys Va - #l Pro Phe Leu Lys                           80 - #                 85 - #                 90              - - Val Pro Ser Ser Pro Ser Ala Trp Leu Asn Se - #r Glu Ala Asn Tyr                           95 - #                100 - #                105              - - Cys Glu Leu Asn Pro Ala Phe Ala Thr Gly Cy - #s Gly Arg Gly Ala                          110  - #               115  - #               120              - - Lys Leu Pro Ser Cys Ala Gln Gly Ser His Th - #r Glu Leu Leu Thr                          125  - #               130  - #               135              - - Ala Lys Gln Asn Glu Ala Pro Gly Pro Arg As - #n Ser Gly Val Asn                          140  - #               145  - #               150              - - Tyr Leu Ile Leu Asp Asp Asp Asp Arg Glu Ar - #g Pro Trp Glu Pro                          155  - #               160  - #               165              - - Ala Ala Ala Gln Met Glu Lys Gly Gln Trp As - #p Lys Gly Glu Phe                          170  - #               175  - #               180              - - Val Thr Pro Leu Leu Glu Thr Val Ser Ser Ph - #e Arg Pro Asn Glu                          185  - #               190  - #               195              - - Phe Glu Ser Lys Phe Leu Pro Pro Glu Asn Ly - #s Pro Leu Glu Thr                          200  - #               205  - #               210              - - Ala Met Leu Lys Arg Ala Lys Glu Leu Phe Th - #r Asn Asn Asp Pro                          215  - #               220  - #               225              - - Lys Val Ile Ala Gln His Val Leu Ser Met As - #p Cys Arg Val Ala                          230  - #               235  - #               240              - - Arg Ile Leu Gly Val Ser Glu Glu Met Arg Ar - #g Asn Met Gly Val                          245  - #               250  - #               255              - - Ser Ser Gly Leu Glu Leu Ile Thr Leu Pro Hi - #s Gly His Gln Leu                          260  - #               265  - #               270              - - Arg Leu Asp Ile Ile Glu Arg His Asn Thr Me - #t Ala Ile Gly Ile                          275  - #               280  - #               285              - - Ala Val Asp Ile Leu Gly Cys Thr Gly Thr Le - #u Glu Asp Arg Ala                          290  - #               295  - #               300              - - Ala Thr Leu Ser Lys Ile Ile Gln Val Ala Va - #l Glu Leu Lys Asp                          305  - #               310  - #               315              - - Ser Met Gly Asp Leu Tyr Ser Phe Ser Ala Le - #u Met Lys Ala Leu                          320  - #               325  - #               330              - - Glu Met Pro Gln Ile Thr Arg Leu Glu Lys Th - #r Trp Thr Ala Leu                          335  - #               340  - #               345              - - Arg His Gln Tyr Thr Gln Thr Ala Ile Leu Ty - #r Glu Lys Gln Leu                          350  - #               355  - #               360              - - Lys Pro Phe Ser Lys Leu Leu His Glu Gly Ar - #g Glu Ser Thr Cys                          365  - #               370  - #               375              - - Val Pro Pro Asn Asn Val Ser Val Pro Leu Le - #u Met Pro Leu Val                          380  - #               385  - #               390              - - Thr Leu Met Glu Arg Gln Ala Val Thr Phe Gl - #u Gly Thr Asp Met                          395  - #               400  - #               405              - - Trp Glu Lys Asn Asp Gln Ser Cys Glu Ile Me - #t Leu Asn His Leu                          410  - #               415  - #               420              - - Ala Thr Ala Arg Phe Met Ala Glu Ala Ala As - #p Ser Tyr Arg Met                          425  - #               430  - #               435              - - Asn Ala Glu Arg Ile Leu Ala Gly Phe Gln Pr - #o Asp Glu Glu Met                          440  - #               445  - #               450              - - Asn Glu Ile Cys Lys Thr Glu Phe Gln Met Ar - #g Leu Leu Trp Gly                          455  - #               460  - #               465              - - Ser Lys Gly Ala Gln Val Asn Gln Thr Glu Ar - #g Tyr Glu Lys Phe                          470  - #               475  - #               480              - - Asn Gln Ile Leu Thr Ala Leu Ser Arg Lys Le - #u Glu Pro Pro Pro                          485  - #               490  - #               495              - - Val Lys Gln Ala Glu Leu                                                                  500 5 - #01                                                    - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 2174                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 4                                                         - - ggcccctgga gtccagccgc agtggtcact gcttaaatat cacttctcgg  - #                  50                                                                         - - gagatatttc cttttgtaat ttgccctcgg tcttgtctta tcttcgaaag  - #                 100                                                                         - - gttgctggaa tttctctgtt ccttggagtt tgggggtttt ttgatttgtt  - #                 150                                                                         - - ttttctttgg tgcttgtaaa gaaacaaaga aaagagtggt agccagcccg  - #                 200                                                                         - - cctgcctgga tcac atg cag gac aga aga gcc ttg t - #cc ctc                    - #  241                                                                                    Met G - #ln Asp Arg Arg Ala Leu Ser Leu                                         - #1               5                                         - - aaa gcc cac cag tca gag agc tac ctg ccg at - #t ggc tgc                  - #    280                                                                    Lys Ala His Gln Ser Glu Ser Tyr Leu Pro Il - #e Gly Cys                        10                 - # 15                 - # 20                              - - aag ctg cca cct cag tcc tcg ggt gtg gac ac - #a agc ccc                  - #    319                                                                    Lys Leu Pro Pro Gln Ser Ser Gly Val Asp Th - #r Ser Pro                                25         - #         30         - #         35                      - - tgc cca aac tca cct gtg ttc agg acg gga ag - #c gag cct                  - #    358                                                                    Cys Pro Asn Ser Pro Val Phe Arg Thr Gly Se - #r Glu Pro                                        40 - #                 45                                     - - gcc ctg agc cca gca gtg gtt cgg agg gtc tc - #c tca gac                  - #    397                                                                    Ala Leu Ser Pro Ala Val Val Arg Arg Val Se - #r Ser Asp                            50             - #     55             - #     60                          - - gcc agg gct ggg gag gcg ctg agg gga tca ga - #c agt caa                  - #    436                                                                    Ala Arg Ala Gly Glu Ala Leu Arg Gly Ser As - #p Ser Gln                                    65     - #             70                                         - - ctg tgc cct aag ccc ccg cct aag ccc tgc aa - #g gtg ccg                  - #    475                                                                    Leu Cys Pro Lys Pro Pro Pro Lys Pro Cys Ly - #s Val Pro                        75                 - # 80                 - # 85                              - - ttc ctc aag gtt ccc tcg tct ccc tct gcc tg - #g ctc aac                  - #    514                                                                    Phe Leu Lys Val Pro Ser Ser Pro Ser Ala Tr - #p Leu Asn                                90         - #         95         - #        100                      - - tca gag gcc aac tac tgt gaa ctg aac cca gc - #g ttt gcc                  - #    553                                                                    Ser Glu Ala Asn Tyr Cys Glu Leu Asn Pro Al - #a Phe Ala                                       105  - #               110                                     - - aca ggc tgc ggc agg gga gca aag cta ccc tc - #a tgt gcc                  - #    592                                                                    Thr Gly Cys Gly Arg Gly Ala Lys Leu Pro Se - #r Cys Ala                           115              - #   120              - #   125                          - - cag gga agc cac aca gaa ctg ctc aca gcc aa - #g cag aat                  - #    631                                                                    Gln Gly Ser His Thr Glu Leu Leu Thr Ala Ly - #s Gln Asn                                   130      - #           135                                         - - gag gcg cca ggt ccc cgg aac tct ggc gtc aa - #c tac ttg                  - #    670                                                                    Glu Ala Pro Gly Pro Arg Asn Ser Gly Val As - #n Tyr Leu                       140                 1 - #45                 1 - #50                            - - atc ctt gat gat gat gac agg gaa aga cct tg - #g gaa cct                  - #    709                                                                    Ile Leu Asp Asp Asp Asp Arg Glu Arg Pro Tr - #p Glu Pro                               155          - #       160          - #       165                      - - gcg gca gct cag atg gag aag ggg cag tgg ga - #c aag ggc                  - #    748                                                                    Ala Ala Ala Gln Met Glu Lys Gly Gln Trp As - #p Lys Gly                                       170  - #               175                                     - - gag ttt gtg acg ccc ctc ctg gag act gtc tc - #c tcc ttc                  - #    787                                                                    Glu Phe Val Thr Pro Leu Leu Glu Thr Val Se - #r Ser Phe                           180              - #   185              - #   190                          - - agg ccc aac gag ttt gag tca aag ttc ctt cc - #c cct gag                  - #    826                                                                    Arg Pro Asn Glu Phe Glu Ser Lys Phe Leu Pr - #o Pro Glu                                   195      - #           200                                         - - aat aag ccc ctg gaa aca gca atg ttg aaa cg - #t gca aaa                  - #    865                                                                    Asn Lys Pro Leu Glu Thr Ala Met Leu Lys Ar - #g Ala Lys                       205                 2 - #10                 2 - #15                            - - gaa ctg ttc acc aac aac gac ccc aag gtc at - #c gcc cag                  - #    904                                                                    Glu Leu Phe Thr Asn Asn Asp Pro Lys Val Il - #e Ala Gln                               220          - #       225          - #       230                      - - cac gta ctg agc atg gac tgc agg gtt gct ag - #g ata ctt                  - #    943                                                                    His Val Leu Ser Met Asp Cys Arg Val Ala Ar - #g Ile Leu                                       235  - #               240                                     - - gga gtc tct gaa gag atg agg agg aac atg gg - #g gtg agc                  - #    982                                                                    Gly Val Ser Glu Glu Met Arg Arg Asn Met Gl - #y Val Ser                           245              - #   250              - #   255                          - - tca ggc ctg gaa ctc att acc ttg cct cac gg - #a cac cag                  - #   1021                                                                    Ser Gly Leu Glu Leu Ile Thr Leu Pro His Gl - #y His Gln                                   260      - #           265                                         - - ctg cgc ctg gac ata att gaa aga cac aac ac - #a atg gcc                  - #   1060                                                                    Leu Arg Leu Asp Ile Ile Glu Arg His Asn Th - #r Met Ala                       270                 2 - #75                 2 - #80                            - - atc ggc att gca gtg gac att ctg gga tgc ac - #g ggc act                  - #   1099                                                                    Ile Gly Ile Ala Val Asp Ile Leu Gly Cys Th - #r Gly Thr                               285          - #       290          - #       295                      - - ttg gag gac cga gcg gcc act ctg agt aag at - #c atc cag                  - #   1138                                                                    Leu Glu Asp Arg Ala Ala Thr Leu Ser Lys Il - #e Ile Gln                                       300  - #               305                                     - - gtg gcg gtg gaa ctg aag gat tcc atg ggg ga - #c ctc tat                  - #   1177                                                                    Val Ala Val Glu Leu Lys Asp Ser Met Gly As - #p Leu Tyr                           310              - #   315              - #   320                          - - tcc ttc tca gct ctc atg aaa gcc ctg gaa at - #g cca cag                  - #   1216                                                                    Ser Phe Ser Ala Leu Met Lys Ala Leu Glu Me - #t Pro Gln                                   325      - #           330                                         - - atc aca agg tta gaa aag acg tgg act gct ct - #g cgg cac                  - #   1255                                                                    Ile Thr Arg Leu Glu Lys Thr Trp Thr Ala Le - #u Arg His                       335                 3 - #40                 3 - #45                            - - cag tac acc caa act gcc att ctc tat gag aa - #a cag ctg                  - #   1294                                                                    Gln Tyr Thr Gln Thr Ala Ile Leu Tyr Glu Ly - #s Gln Leu                               350          - #       355          - #       360                      - - aag ccc ttc agc aaa ctc ctg cat gaa ggc ag - #a gag tcc                  - #   1333                                                                    Lys Pro Phe Ser Lys Leu Leu His Glu Gly Ar - #g Glu Ser                                       365  - #               370                                     - - aca tgt gtt ccc cca aac aat gta tca gtc cc - #a ctg ctg                  - #   1372                                                                    Thr Cys Val Pro Pro Asn Asn Val Ser Val Pr - #o Leu Leu                           375              - #   380              - #   385                          - - atg ccg ctt gtg acg tta atg gag cgc cag gc - #t gtg act                  - #   1411                                                                    Met Pro Leu Val Thr Leu Met Glu Arg Gln Al - #a Val Thr                                   390      - #           395                                         - - ttt gaa gga acc gac atg tgg gaa aaa aac ga - #c cag agc                  - #   1450                                                                    Phe Glu Gly Thr Asp Met Trp Glu Lys Asn As - #p Gln Ser                       400                 4 - #05                 4 - #10                            - - tgt gaa atc atg ctg aac cat ttg gca aca gc - #g cga ttc                  - #   1489                                                                    Cys Glu Ile Met Leu Asn His Leu Ala Thr Al - #a Arg Phe                               415          - #       420          - #       425                      - - atg gcc gag gct gca gac agc tac cgg atg aa - #t gct gag                  - #   1528                                                                    Met Ala Glu Ala Ala Asp Ser Tyr Arg Met As - #n Ala Glu                                       430  - #               435                                     - - agg atc ctg gca ggt ttt caa cca gat gaa ga - #a atg aat                  - #   1567                                                                    Arg Ile Leu Ala Gly Phe Gln Pro Asp Glu Gl - #u Met Asn                           440              - #   445              - #   450                          - - gaa atc tgc aag act gaa ttt caa atg cga tt - #g cta tgg                  - #   1606                                                                    Glu Ile Cys Lys Thr Glu Phe Gln Met Arg Le - #u Leu Trp                                   455      - #           460                                         - - ggc agc aaa ggt gca caa gtc aat cag aca ga - #g aga tat                  - #   1645                                                                    Gly Ser Lys Gly Ala Gln Val Asn Gln Thr Gl - #u Arg Tyr                       465                 4 - #70                 4 - #75                            - - gag aaa ttc aac cag att tta act gcc ctc tc - #g cgt aaa                  - #   1684                                                                    Glu Lys Phe Asn Gln Ile Leu Thr Ala Leu Se - #r Arg Lys                               480          - #       485          - #       490                      - - ttg gaa cct cct cct gta aag cag gca gag ct - #t tga                     - #     1720                                                                    Leu Glu Pro Pro Pro Val Lys Gln Ala Glu Le - #u                                               495  - #               500 501                                 - - taactctcca gagaaccttt agaatatctt ttcaagtttc cccagcttca  - #                1770                                                                        - - tctttgggaa agcttactgt ttttgataaa gtaataatgt gcaaatctga  - #                1820                                                                         - - caatatacaa gcttttagta tccacaggat attaaacgtg taaattgcac  - #                1870                                                                         - - agagcacact tatttatgaa ttgtctaaag ttactactga ttttaaaatg  - #                1920                                                                         - - aataatttat tattaaggta actactgcta atgttgatca gcaaatttaa  - #                1970                                                                         - - gagaagacct agctatgttg gctggttgct ttctattatc atggtatttg  - #                2020                                                                         - - accattttag ttttaattcc atgtcagata agtgtaaata gaagagttta  - #                2070                                                                         - - aaagcatgaa acatttcaga aggtatcagt tatatgatat tctttaaaca  - #                2120                                                                         - - aatatgaaaa atgtaaatac tcatgaatga aaatacatct ttttgtgaaa  - #                2170                                                                         - - cagt                 - #                  - #                  - #               2174                                                                   - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 703                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 5                                                         - - Met Thr Ala Val Gly Arg Arg Cys Pro Ala Le - #u Gly Ser Arg Gly            1               5 - #                 10 - #                 15              - - Ala Ala Gly Glu Pro Glu Ala Gly Ser Asp Ty - #r Val Lys Phe Ser                           20 - #                 25 - #                 30              - - Lys Glu Lys Tyr Ile Leu Asp Ser Ser Pro Gl - #u Lys Leu His Lys                           35 - #                 40 - #                 45              - - Glu Leu Glu Glu Glu Leu Lys Leu Ser Ser Th - #r Asp Leu Arg Ser                           50 - #                 55 - #                 60              - - His Ala Trp Tyr His Gly Arg Ile Pro Arg Gl - #u Val Ser Glu Thr                           65 - #                 70 - #                 75              - - Leu Val Gln Arg Asn Gly Asp Phe Leu Ile Ar - #g Asp Ser Leu Thr                           80 - #                 85 - #                 90              - - Ser Leu Gly Asp Tyr Val Leu Thr Cys Arg Tr - #p Arg Asn Gln Ala                           95 - #                100 - #                105              - - Leu His Phe Lys Ile Asn Lys Val Val Val Ly - #s Ala Gly Glu Ser                          110  - #               115  - #               120              - - Tyr Thr His Ile Gln Tyr Leu Phe Glu Gln Gl - #u Ser Phe Asp His                          125  - #               130  - #               135              - - Val Pro Ala Leu Val Arg Tyr His Val Gly Se - #r Arg Lys Ala Val                          140  - #               145  - #               150              - - Ser Glu Gln Ser Gly Ala Ile Ile Tyr Cys Pr - #o Val Asn Arg Thr                          155  - #               160  - #               165              - - Phe Pro Leu Arg Tyr Leu Glu Ala Ser Tyr Gl - #y Leu Gly Gln Gly                          170  - #               175  - #               180              - - Ser Ser Lys Pro Ala Ser Pro Val Ser Pro Se - #r Gly Pro Lys Gly                          185  - #               190  - #               195              - - Ser His Met Lys Arg Arg Ser Val Thr Met Th - #r Asp Gly Leu Thr                          200  - #               205  - #               210              - - Ala Asp Lys Val Thr Arg Ser Asp Gly Cys Pr - #o Thr Ser Thr Ser                          215  - #               220  - #               225              - - Leu Pro Arg Pro Arg Asp Ser Ile Arg Ser Cy - #s Ala Leu Ser Met                          230  - #               235  - #               240              - - Asp Gln Ile Pro Asp Leu His Ser Pro Met Se - #r Pro Ile Ser Glu                          245  - #               250  - #               255              - - Ser Pro Ser Ser Pro Ala Tyr Ser Thr Val Th - #r Arg Val His Ala                          260  - #               265  - #               270              - - Ala Pro Ala Ala Pro Ser Ala Thr Ala Leu Pr - #o Ala Ser Pro Val                          275  - #               280  - #               285              - - Ala Arg Cys Ser Ser Glu Pro Gln Leu Cys Pr - #o Gly Ser Ala Pro                          290  - #               295  - #               300              - - Lys Thr His Gly Glu Ser Asp Lys Gly Pro Hi - #s Thr Ser Pro Ser                          305  - #               310  - #               315              - - His Thr Leu Gly Lys Ala Ser Pro Ser Pro Se - #r Leu Ser Ser Tyr                          320  - #               325  - #               330              - - Ser Asp Pro Asp Ser Gly His Tyr Cys Gln Le - #u Gln Pro Pro Val                          335  - #               340  - #               345              - - Arg Gly Ser Arg Glu Trp Ala Ala Thr Glu Th - #r Ser Ser Gln Gln                          350  - #               355  - #               360              - - Ala Arg Ser Tyr Gly Glu Arg Leu Lys Glu Le - #u Ser Glu Asn Gly                          365  - #               370  - #               375              - - Ala Pro Glu Gly Asp Trp Gly Lys Thr Phe Th - #r Val Pro Ile Val                          380  - #               385  - #               390              - - Glu Val Thr Ser Ser Phe Asn Pro Ala Thr Ph - #e Gln Ser Leu Leu                          395  - #               400  - #               405              - - Ile Pro Arg Asp Asn Arg Pro Leu Glu Val Gl - #y Leu Leu Arg Lys                          410  - #               415  - #               420              - - Val Lys Glu Leu Leu Ala Glu Val Asp Ala Ar - #g Thr Leu Ala Arg                          425  - #               430  - #               435              - - His Val Thr Lys Val Asp Cys Leu Val Ala Ar - #g Ile Leu Gly Val                          440  - #               445  - #               450              - - Thr Lys Glu Met Gln Thr Leu Met Gly Val Ar - #g Trp Gly Met Glu                          455  - #               460  - #               465              - - Leu Leu Thr Leu Pro His Gly Arg Gln Leu Ar - #g Leu Asp Leu Leu                          470  - #               475  - #               480              - - Glu Arg Phe His Thr Met Ser Ile Met Leu Al - #a Val Asp Ile Leu                          485  - #               490  - #               495              - - Gly Cys Thr Gly Ser Ala Glu Glu Arg Ala Al - #a Leu Leu His Lys                          500  - #               505  - #               510              - - Thr Ile Gln Leu Ala Ala Glu Leu Arg Gly Th - #r Met Gly Asn Met                          515  - #               520  - #               525              - - Phe Ser Phe Ala Ala Val Met Gly Ala Leu As - #p Met Ala Gln Ile                          530  - #               535  - #               540              - - Ser Arg Leu Glu Gln Thr Trp Val Thr Leu Ar - #g Gln Arg His Thr                          545  - #               550  - #               555              - - Glu Gly Ala Ile Leu Tyr Glu Lys Lys Leu Ly - #s Pro Phe Leu Lys                          560  - #               565  - #               570              - - Ser Leu Asn Glu Gly Lys Glu Gly Pro Pro Le - #u Ser Asn Thr Thr                          575  - #               580  - #               585              - - Phe Pro His Val Leu Pro Leu Ile Thr Leu Le - #u Glu Cys Asp Ser                          590  - #               595  - #               600              - - Ala Pro Pro Glu Gly Pro Glu Pro Trp Gly Se - #r Thr Glu His Gly                          605  - #               610  - #               615              - - Val Glu Val Val Leu Ala His Leu Glu Ala Al - #a Arg Thr Val Ala                          620  - #               625  - #               630              - - His His Gly Gly Leu Tyr His Thr Asn Ala Gl - #u Val Lys Leu Gln                          635  - #               640  - #               645              - - Gly Phe Gln Ala Arg Pro Glu Leu Leu Glu Va - #l Phe Ser Thr Glu                          650  - #               655  - #               660              - - Phe Gln Met Arg Leu Leu Trp Gly Ser Gln Gl - #y Ala Ser Ser Ser                          665  - #               670  - #               675              - - Gln Ala Arg Arg Tyr Glu Lys Phe Asp Lys Va - #l Leu Thr Ala Leu                          680  - #               685  - #               690              - - Ser His Lys Leu Glu Pro Ala Val Arg Ser Se - #r Glu Leu                                  695  - #               700  - #       703                      - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 2153                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 6                                                         - - taggaggtcc ccgggttgcc ggcggcgaca gcgggggaag c   atg - #                      - # 44                                                                                      - #                  - #            Met                                       - #                  - #              1                      - - act gct gtg ggc cga agg tgc ccc gcg ctg gg - #g tcc cga                  - #     83                                                                    Thr Ala Val Gly Arg Arg Cys Pro Ala Leu Gl - #y Ser Arg                                     5    - #              10                                         - - ggg gct gct gga gag cca gag gct ggc agc ga - #c tat gtg                  - #    122                                                                    Gly Ala Ala Gly Glu Pro Glu Ala Gly Ser As - #p Tyr Val                        15                 - # 20                 - # 25                              - - aag ttc tcc aag gag aag tac atc ctg gac tc - #a tcg cca                  - #    161                                                                    Lys Phe Ser Lys Glu Lys Tyr Ile Leu Asp Se - #r Ser Pro                                30         - #         35         - #         40                      - - gag aaa ctc cac aag gaa ttg gag gag gag ct - #c aaa ctc                  - #    200                                                                    Glu Lys Leu His Lys Glu Leu Glu Glu Glu Le - #u Lys Leu                                        45 - #                 50                                     - - agc agc acg gat ctc cgc agc cat gcc tgg ta - #c cat ggc                  - #    239                                                                    Ser Ser Thr Asp Leu Arg Ser His Ala Trp Ty - #r His Gly                            55             - #     60             - #     65                          - - cgc atc ccc cga gag gtc tcg gag acc ttg gt - #a caa cgc                  - #    278                                                                    Arg Ile Pro Arg Glu Val Ser Glu Thr Leu Va - #l Gln Arg                                    70     - #             75                                         - - aac ggc gac ttc ctc atc cgg gac tcg ctc ac - #c agc ctg                  - #    317                                                                    Asn Gly Asp Phe Leu Ile Arg Asp Ser Leu Th - #r Ser Leu                        80                 - # 85                 - # 90                              - - ggc gac tat gtg ctc acg tgc cgc tgg cgc aa - #c cag gcc                  - #    356                                                                    Gly Asp Tyr Val Leu Thr Cys Arg Trp Arg As - #n Gln Ala                                95         - #        100         - #        105                      - - ttg cac ttc aag atc aac aag gtg gtg gtg aa - #g gca ggc                  - #    395                                                                    Leu His Phe Lys Ile Asn Lys Val Val Val Ly - #s Ala Gly                                       110  - #               115                                     - - gag agc tac aca cac atc cag tac ctg ttt ga - #g cag gag                  - #    434                                                                    Glu Ser Tyr Thr His Ile Gln Tyr Leu Phe Gl - #u Gln Glu                           120              - #   125              - #   130                          - - agc ttt gac cac gtg ccc gcc ctc gtg cgc ta - #t cat gtg                  - #    473                                                                    Ser Phe Asp His Val Pro Ala Leu Val Arg Ty - #r His Val                                   135      - #           140                                         - - ggc agc cgc aag gct gtg tca gag cag agt gg - #t gcc atc                  - #    512                                                                    Gly Ser Arg Lys Ala Val Ser Glu Gln Ser Gl - #y Ala Ile                       145                 1 - #50                 1 - #55                            - - atc tac tgc ccg gtg aac cgc acc ttc cca ct - #g cgc tac                  - #    551                                                                    Ile Tyr Cys Pro Val Asn Arg Thr Phe Pro Le - #u Arg Tyr                               160          - #       165          - #       170                      - - ctc gag gcc agc tat ggc ctg gga cag ggg ag - #t agc aag                  - #    590                                                                    Leu Glu Ala Ser Tyr Gly Leu Gly Gln Gly Se - #r Ser Lys                                       175  - #               180                                     - - cct gct agc ccc gtc agc ccc tca ggc ccc aa - #g ggc agc                  - #    629                                                                    Pro Ala Ser Pro Val Ser Pro Ser Gly Pro Ly - #s Gly Ser                           185              - #   190              - #   195                          - - cac atg aag cgg cgc agc gtc acc atg acc ga - #t ggg ctc                  - #    668                                                                    His Met Lys Arg Arg Ser Val Thr Met Thr As - #p Gly Leu                                   200      - #           205                                         - - act gct gac aag gtc acc cgc agc gat ggc tg - #c ccc acc                  - #    707                                                                    Thr Ala Asp Lys Val Thr Arg Ser Asp Gly Cy - #s Pro Thr                       210                 2 - #15                 2 - #20                            - - agt acg tcg ctg ccc cgc cct cgg gac tcc at - #c cgc agc                  - #    746                                                                    Ser Thr Ser Leu Pro Arg Pro Arg Asp Ser Il - #e Arg Ser                               225          - #       230          - #       235                      - - tgt gcc ctc agc atg gac cag atc cca gac ct - #g cac tca                  - #    785                                                                    Cys Ala Leu Ser Met Asp Gln Ile Pro Asp Le - #u His Ser                                       240  - #               245                                     - - ccc atg tcg ccc atc tcc gag agc cct agc tc - #c cct gcc                  - #    824                                                                    Pro Met Ser Pro Ile Ser Glu Ser Pro Ser Se - #r Pro Ala                           250              - #   255              - #   260                          - - tac agc act gta acc cgt gtc cat gcc gcc cc - #t gca gcc                  - #    863                                                                    Tyr Ser Thr Val Thr Arg Val His Ala Ala Pr - #o Ala Ala                                   265      - #           270                                         - - cct tct gcc aca gca ttg cct gcc tcc cct gt - #c gcc cgc                  - #    902                                                                    Pro Ser Ala Thr Ala Leu Pro Ala Ser Pro Va - #l Ala Arg                       275                 2 - #80                 2 - #85                            - - tgt tcc agt gag ccc cag ctg tgt ccc gga ag - #t gcc cca                  - #    941                                                                    Cys Ser Ser Glu Pro Gln Leu Cys Pro Gly Se - #r Ala Pro                               290          - #       295          - #       300                      - - aag acc cat ggg gag tca gac aag ggc ccc ca - #c acc agc                  - #    980                                                                    Lys Thr His Gly Glu Ser Asp Lys Gly Pro Hi - #s Thr Ser                                       305  - #               310                                     - - ccc tcc cac acc ctt ggc aag gcc tcc ccg tc - #a cca tca                  - #   1019                                                                    Pro Ser His Thr Leu Gly Lys Ala Ser Pro Se - #r Pro Ser                           315              - #   320              - #   325                          - - ctc agc agc tac agt gac ccg gac tct ggc ca - #c tac tgc                  - #   1058                                                                    Leu Ser Ser Tyr Ser Asp Pro Asp Ser Gly Hi - #s Tyr Cys                                   330      - #           335                                         - - cag ctc cag cct ccc gtg cgt ggc agc cga ga - #g tgg gca                  - #   1097                                                                    Gln Leu Gln Pro Pro Val Arg Gly Ser Arg Gl - #u Trp Ala                       340                 3 - #45                 3 - #50                            - - gcg act gag acc tcc agc cag cag gcc agg ag - #c tat ggg                  - #   1136                                                                    Ala Thr Glu Thr Ser Ser Gln Gln Ala Arg Se - #r Tyr Gly                               355          - #       360          - #       365                      - - gag agg cta aag gaa ctg tca gaa aat ggg gc - #c cct gaa                  - #   1175                                                                    Glu Arg Leu Lys Glu Leu Ser Glu Asn Gly Al - #a Pro Glu                                       370  - #               375                                     - - ggg gac tgg ggc aag acc ttc aca gtc ccc at - #c gtg gaa                  - #   1214                                                                    Gly Asp Trp Gly Lys Thr Phe Thr Val Pro Il - #e Val Glu                           380              - #   385              - #   390                          - - gtc act tct tcc ttc aac ccg gcc acc ttc ca - #g tca cta                  - #   1253                                                                    Val Thr Ser Ser Phe Asn Pro Ala Thr Phe Gl - #n Ser Leu                                   395      - #           400                                         - - ctg atc ccc agg gat aac cgg cca ctg gag gt - #g ggc ctt                  - #   1292                                                                    Leu Ile Pro Arg Asp Asn Arg Pro Leu Glu Va - #l Gly Leu                       405                 4 - #10                 4 - #15                            - - ctg cgc aag gtc aag gag ctg ctg gca gaa gt - #g gat gcc                  - #   1331                                                                    Leu Arg Lys Val Lys Glu Leu Leu Ala Glu Va - #l Asp Ala                               420          - #       425          - #       430                      - - cgg acg ctg gcc cgg cat gtc acc aag gtg ga - #c tgc ctg                  - #   1370                                                                    Arg Thr Leu Ala Arg His Val Thr Lys Val As - #p Cys Leu                                       435  - #               440                                     - - gtt gct agg ata ctg ggc gtt acc aag gag at - #g cag acc                  - #   1409                                                                    Val Ala Arg Ile Leu Gly Val Thr Lys Glu Me - #t Gln Thr                           445              - #   450              - #   455                          - - cta atg gga gtc cgc tgg ggc atg gaa ctg ct - #c acc ctc                  - #   1448                                                                    Leu Met Gly Val Arg Trp Gly Met Glu Leu Le - #u Thr Leu                                   460      - #           465                                         - - ccc cat ggc cgg cag cta cgc cta gac ctg ct - #g gaa agg                  - #   1487                                                                    Pro His Gly Arg Gln Leu Arg Leu Asp Leu Le - #u Glu Arg                       470                 4 - #75                 4 - #80                            - - ttc cac acc atg tcc atc atg ctg gcc gtg ga - #c atc ctg                  - #   1526                                                                    Phe His Thr Met Ser Ile Met Leu Ala Val As - #p Ile Leu                               485          - #       490          - #       495                      - - ggc tgc acc ggc tct gcg gag gag cgg gca gc - #g ctg ctg                  - #   1565                                                                    Gly Cys Thr Gly Ser Ala Glu Glu Arg Ala Al - #a Leu Leu                                       500  - #               505                                     - - cac aag acc att cag ctg gcg gcc gag cta cg - #g ggg act                  - #   1604                                                                    His Lys Thr Ile Gln Leu Ala Ala Glu Leu Ar - #g Gly Thr                           510              - #   515              - #   520                          - - atg ggc aac atg ttc agc ttc gcg gcg gtc at - #g ggt gcc                  - #   1643                                                                    Met Gly Asn Met Phe Ser Phe Ala Ala Val Me - #t Gly Ala                                   525      - #           530                                         - - ctg gac atg gct cag att tct cgg ctg gag ca - #g aca tgg                  - #   1682                                                                    Leu Asp Met Ala Gln Ile Ser Arg Leu Glu Gl - #n Thr Trp                       535                 5 - #40                 5 - #45                            - - gtg acc ctg cgg cag cga cac aca gag ggt gc - #c atc ctg                  - #   1721                                                                    Val Thr Leu Arg Gln Arg His Thr Glu Gly Al - #a Ile Leu                               550          - #       555          - #       560                      - - tac gag aag aag ctc aag cct ttt ctc aag ag - #c ctc aac                  - #   1760                                                                    Tyr Glu Lys Lys Leu Lys Pro Phe Leu Lys Se - #r Leu Asn                                       565  - #               570                                     - - gag ggc aaa gaa ggc ccg ccg ctg agc aac ac - #c acg ttt                  - #   1799                                                                    Glu Gly Lys Glu Gly Pro Pro Leu Ser Asn Th - #r Thr Phe                           575              - #   580              - #   585                          - - cct cat gtg ctg ccc ctc atc acc ctg ctg ga - #g tgt gac                  - #   1838                                                                    Pro His Val Leu Pro Leu Ile Thr Leu Leu Gl - #u Cys Asp                                   590      - #           595                                         - - tcg gcc cca cca gag ggc cct gag ccc tgg gg - #c agc acg                  - #   1877                                                                    Ser Ala Pro Pro Glu Gly Pro Glu Pro Trp Gl - #y Ser Thr                       600                 6 - #05                 6 - #10                            - - gag cac ggc gtg gag gtg gtg ctg gct cac ct - #g gag gcc                  - #   1916                                                                    Glu His Gly Val Glu Val Val Leu Ala His Le - #u Glu Ala                               615          - #       620          - #       625                      - - gcc cgc aca gtg gca cac cac gga ggc ctg ta - #c cac acc                  - #   1955                                                                    Ala Arg Thr Val Ala His His Gly Gly Leu Ty - #r His Thr                                       630  - #               635                                     - - aat gct gaa gtc aag ctg cag ggg ttc cag gc - #c cgg ccg                  - #   1994                                                                    Asn Ala Glu Val Lys Leu Gln Gly Phe Gln Al - #a Arg Pro                           640              - #   645              - #   650                          - - gag ctc ctg gag gtg ttc agc acg gag ttc ca - #g atg cgc                  - #   2033                                                                    Glu Leu Leu Glu Val Phe Ser Thr Glu Phe Gl - #n Met Arg                                   655      - #           660                                         - - ctt ctc tgg ggc agt cag ggt gcc agc agc ag - #c cag gcc                  - #   2072                                                                    Leu Leu Trp Gly Ser Gln Gly Ala Ser Ser Se - #r Gln Ala                       665                 6 - #70                 6 - #75                            - - cgg cgc tat gag aag ttc gac aag gtc ctc ac - #t gcc ctg                  - #   2111                                                                    Arg Arg Tyr Glu Lys Phe Asp Lys Val Leu Th - #r Ala Leu                               680          - #       685          - #       690                      - - tcc cac aag ctg gaa cct gct gtc cgc tcc ag - #c gag ctg                  - #   2150                                                                    Ser His Lys Leu Glu Pro Ala Val Arg Ser Se - #r Glu Leu                                       695  - #               700  - #       703                      - - tga                  - #                  - #                  - #               2153                                                                   - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 30                                                              <212> TYPE: DNA                                                               <213> ORGANISM: artificial                                                    <220> FEATURE:                                                                <221> NAME/KEY: artificial sequence                                           <222> LOCATION: 1-30                                                          <223> OTHER INFORMATION: artificial sequence                                   - - <400> SEQUENCE: 7                                                         - - actgaggcct gttgaaagtg cagagctcag         - #                  - #               30                                                                      - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: artifical                                                     <220> FEATURE:                                                                <221> NAME/KEY: artificial sequence                                           <222> LOCATION: 1-18                                                          <223> OTHER INFORMATION: artificial sequence                                   - - <400> SEQUENCE: 8                                                         - - ctgaagaag agcttcag             - #                  - #                      - #   18                                                                   - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: artificial                                                    <220> FEATURE:                                                                <221> NAME/KEY: artificial sequence                                           <222> LOCATION: 1-48                                                          <223> OTHER INFORMATION: artificial sequence                                   - - <400> SEQUENCE: 9                                                         - - caatgccgat ggccattgtg ttgtgtcttt caattatgtc caggcgca  - #                    48                                                                         - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: artificial                                                    <220> FEATURE:                                                                <221> NAME/KEY: artificial sequence                                           <222> LOCATION: 1-18                                                          <223> OTHER INFORMATION: artificial sequence                                   - - <400> SEQUENCE: 10                                                        - - atcccagaat gtccactg             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 50                                                              <212> TYPE: DNA                                                               <213> ORGANISM: artificial                                                    <220> FEATURE:                                                                <221> NAME/KEY: artificial sequence                                           <222> LOCATION: 1-50                                                          <223> OTHER INFORMATION: artificial sequence                                   - - <400> SEQUENCE: 11                                                        - - ggccagcatg atggacatgg tgtggaacct ttccagcagg tctaggcgta  - #                  50                                                                         - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: artificial                                                    <220> FEATURE:                                                                <221> NAME/KEY: artificial sequence                                           <222> LOCATION: 1-18                                                          <223> OTHER INFORMATION: artificial sequence                                   - - <400> SEQUENCE: 12                                                        - - ggtgcagccc aggatgtc             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 254                                                             <212> TYPE: DNA                                                               <213> ORGANISM: unknown                                                       <220> FEATURE:                                                                <221> NAME/KEY: unknown source                                                <222> LOCATION: 1-254                                                         <223> OTHER INFORMATION:                                                       - - <400> SEQUENCE: 13                                                        - - gtggagggcg ggggtgacag cagcccggag ccgcggagcc tcagcttccg  - #                  50                                                                         - - cctggaccca gcctcgtggg agccccgcgg gtcctgccca gatgtggaag  - #                 100                                                                         - - actgaggcct gttgaaagtg cagagctcag ccctggcacc ctctgttccc  - #                 150                                                                         - - aagagctcca tgcaggtgcc acaggatgga gaagaccttg ctggccaacc  - #                 200                                                                         - - ttggtaccac ggcctcctgt cccgccagaa ggctgaagct cttcttcagc  - #                 250                                                                         - - aaaa                 - #                  - #                  - #                254                                                                   - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 212                                                             <212> TYPE: DNA                                                               <213> ORGANISM: unknown                                                       <220> FEATURE:                                                                <221> NAME/KEY: unknown N                                                     <222> LOCATION: 59                                                            <223> OTHER INFORMATION:                                                      <220> FEATURE:                                                                <221> NAME/KEY: unknown source                                                <222> LOCATION: 1-212                                                         <223> OTHER INFORMATION:                                                       - - <400> SEQUENCE: 14                                                        - - catcgcccag cacgtactga gcatggactg cagggttgct aggatacttg  - #                  50                                                                         - - gagtctctna agagatgagg aggaacatgg gggtgagctc aggcctggaa  - #                 100                                                                         - - ctcattacct tgcctcacgg acaccagctg cgcctggaca taattgaaag  - #                 150                                                                         - - acacaacaca atggccatcg gcattgcagt ggacattctg ggatgcacgg  - #                 200                                                                         - - gcactttgga gg              - #                  - #                      - #      212                                                                   - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 242                                                             <212> TYPE: DNA                                                               <213> ORGANISM: unknown source                                                <220> FEATURE:                                                                <221> NAME/KEY: unknown N                                                     <222> LOCATION: 204                                                           <223> OTHER INFORMATION:                                                      <220> FEATURE:                                                                <221> NAME/KEY: unknown source                                                <222> LOCATION: 1-242                                                         <223> OTHER INFORMATION:                                                       - - <400> SEQUENCE: 15                                                        - - gctggcagaa gtggatgccc ggacgctggc ccggcatgtc accaaggtgg  - #                  50                                                                         - - actgcctggt tgctaggata ctgggcgtta ccaaggagat gcagacccta  - #                 100                                                                         - - atgggagtcc gctggggcat ggaactgctc accctccccc atggccggca  - #                 150                                                                         - - gctacgccta gacctgctgg aaaggttcca caccatgtcc atcatgctgg  - #                 200                                                                         - - ccgnggacat cctgggctgc accggctctg cggaggagcg gg    - #                      - # 242                                                                      - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 99                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 16                                                        - - Glu Gln Leu Arg Gly Glu Pro Trp Phe His Gl - #y Lys Leu Ser Arg            1               5 - #                 10 - #                 15              - - Arg Glu Ala Glu Ala Leu Leu Gln Leu Asn Gl - #y Asp Phe Leu Val                           20 - #                 25 - #                 30              - - Arg Glu Ser Thr Thr Thr Pro Gly Gln Tyr Va - #l Gly Leu Gln Ser                           35 - #                 40 - #                 45              - - Gly Gln Pro Lys His Leu Leu Leu Val Asp Pr - #o Glu Gly Val Val                           50 - #                 55 - #                 60              - - Arg Thr Lys Asp His Arg Phe Glu Ser Val Se - #r His Leu Ile Ser                           65 - #                 70 - #                 75              - - Tyr His Met Asp Asn Pro Ile Ile Ser Ala Gl - #y Ser Glu Leu Cys                           80 - #                 85 - #                 90              - - Leu Gln Gln Pro Val Glu Arg Lys Leu                                                       95 - #             99                                         - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 99                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 17                                                        - - Glu Gln Leu Arg Gln Glu Pro Trp Tyr His Gl - #y Arg Met Ser Arg            1               5 - #                 10 - #                 15              - - Arg Ala Ala Glu Arg Met Leu Arg Ala Asp Gl - #y Asp Phe Leu Val                           20 - #                 25 - #                 30              - - Arg Asp Ser Val Thr Asn Pro Gly Gln Tyr Va - #l Gly Met His Ala                           35 - #                 40 - #                 45              - - Gly Gln Pro Lys His Leu Leu Leu Val Asp Pr - #o Glu Gly Val Val                           50 - #                 55 - #                 60              - - Arg Thr Lys Asp Val Leu Phe Glu Ser Ile Se - #r His Leu Ile Asp                           65 - #                 70 - #                 75              - - His His Leu Gln Asn Pro Ile Val Ala Ala Gl - #u Ser Glu Leu His                           80 - #                 85 - #                 90              - - Leu Arg Gly Val Val Ser Arg Glu Pro                                                       95 - #             99                                         - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 115                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 18                                                        - - Lys Pro Leu His Glu Gln Leu Trp Tyr His Gl - #y Ala Ile Pro Arg            1               5 - #                 10 - #                 15              - - Ala Glu Val Ala Glu Leu Leu Val His Ser Gl - #y Asp Phe Leu Val                           20 - #                 25 - #                 30              - - Arg Glu Ser Gln Gly Lys Gln Glu Tyr Val Va - #l Leu Trp Asp Gly                           35 - #                 40 - #                 45              - - Leu Pro Arg His Phe Ile Ile Gln Ser Leu As - #p Asn Leu Tyr Arg                           50 - #                 55 - #                 60              - - Leu Glu Gly Glu Gly Phe Pro Ser Ile Pro Le - #u Leu Ile Asp His                           65 - #                 70 - #                 75              - - Leu Leu Ser Thr Pro Leu Thr Lys Lys Ser Gl - #y Val Val Leu His                           80 - #                 85 - #                 90              - - Arg Ala Val Pro Lys Asp Lys Trp Val Leu As - #n His Glu Asp Leu                           95 - #                100 - #                105              - - Val Leu Gly Glu Gln Ile Gly Arg Gly Asn                                                  110  - #               115                                     - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 8                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 19                                                        - - Asp Tyr Lys Asp Asp Asp Asp Lys                                            1               5 - #          8                                           __________________________________________________________________________

What is claimed is:
 1. Isolated nucleic acid comprising DNA having atleast a 95% sequence identity to (a) a DNA molecule encoding a PRO201polypeptide comprising the sequence of amino acids 1 to 576 of FIG. 1(SEQ ID NO: 1), or (b) the complement of the DNA molecule of (a) whereinpercent identity is determined using BLAST-2 software with the defaultparameters.
 2. Isolated nucleic acid comprising DNA having at least a95% sequence identity to (a) a DNA molecule encoding a PRO308polypeptide comprising the sequence of amino acids 1 to 501 of FIG. 2(SEQ ID NO: 3), or (b) the complement of the DNA molecule of (a) whereinpercent identity is determined using BLAST-2 software with the defaultparameters.
 3. The isolated nucleic acid of claim 1 comprising DNAhaving at least a 95% sequence identity to (a) a DNA molecule encoding aPRO309 polypeptide comprising the sequence of amino acids 1 to 703 ofFIG. 3 (SEQ ID NO: 5), or (b) the complement of the DNA molecule of (a)wherein percent identity is determined using BLAST-2 software with thedefault parameters.
 4. The isolated nucleic acid of claim 1 comprisingDNA encoding a PRO201 polypeptide having amino acid residues 1 to 576 ofFIG. 1 (SEQ ID NO: 1).
 5. The isolated nucleic acid of claim 1comprising DNA encoding a PRO308 polypeptide having amino acid residues1 to 501 of FIG. 2 (SEQ ID NO: 3).
 6. The isolated nucleic acid of claim1 comprising DNA encoding a PRO309 polypeptide having amino acidresidues 1 to 703 of FIG. 3 (SEQ ID NO: 5).
 7. An isolated nucleic acidcomprising DNA having at least a 95% sequence identity to (a) a DNAmolecule encoding the same mature polypeptide encoded by the cDNA inATCC Deposit No. 209567 (designation: DNA30676-1223), or (b) thecomplement of the DNA molecule of (a) wherein percent identity isdetermined using BLAST-2 software with the default parameters.
 8. Anisolated nucleic acid comprising DNA having at least a 95% sequenceidentity to (a) a DNA molecule encoding the same mature polypeptideencoded by the cDNA in ATCC Deposit No. 209565 (designation:DNA40575-1223), or (b) the complement of the DNA molecule of (a) whereinpercent identity is determined using BLAST-2 software with the defaultparameters.
 9. An isolated nucleic acid comprising DNA having at least a95% sequence identity to (a) a DNA molecule encoding the same maturepolypeptide encoded by the cDNA in ATCC Deposit No. 209713, or (b) thecomplement of the DNA molecule of (a) wherein percent identity isdetermined using BLAST-2 software with the default parameters.
 10. Avector comprising the nucleic acid of claim
 1. 11. A vector comprisingthe nucleic acid of claim
 2. 12. A vector comprising the nucleic acid ofclaim
 3. 13. The vector of claim 10 operably linked to control sequencesrecognized by a host cell transformed with the vector.
 14. The vector ofclaim 11 operably linked to control sequences recognized by a host celltransformed with the vector.
 15. The vector of claim 12 operably linkedto control sequences recognized by a host cell transformed with thevector.
 16. A host cell comprising the vector of claim
 13. 17. A hostcell comprising the vector of claim
 14. 18. A host cell comprising thevector of claim
 15. 19. The host cell of claim 16 wherein said cell ismammalian.
 20. The host cell of claim 19 wherein said cell is a CHOcell.
 21. The host cell of claim 17 wherein said cell is prokaryotic.22. The host cell of claim 21 wherein said cell is an E. coli.
 23. Thehost cell of claim 18 wherein said cell is a yeast cell.
 24. The hostcell of claim 23 wherein said cell is Saccharomyces cerevisiae.
 25. Aprocess for producing PRO201, PRO308 or PRO309 polypeptides comprisingculturing the host cell of claim 16, 17, or 18 under conditions suitablefor expression of PRO201, PRO308 or PRO309, respectively, and recoveringPRO201, PRO308 or PRO309, respectively, from the cell culture.